WO2015115544A1 - Evaluation method for risk of metastasis or recurrence of colon cancer - Google Patents

Abstract

Provided is a method for objectively and rapidly predicting the risk of metastasis or recurrence of colon cancer with high precision. This method includes a step for measuring expression levels in one or more types of expression products in DNA including transcription initiation regions for biological samples derived from cancerous tissue isolated from colon cancer patients, and evaluates the risk of metastasis or recurrence of colon cancer in those patients.

Description

Evaluation method for colorectal cancer metastasis or recurrence risk

(Related applications and incorporation by reference)
This application claims the priority of Japanese Patent Application No. 2014-017945 filed on January 31, 2014, the entire contents of which are incorporated herein by reference.
Also, all documents cited herein are incorporated by reference in their entirety for all purposes. Citation of any document should not be construed as an admission that it is prior art with respect to the present invention.

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.

For post-surgery treatment of colorectal cancer that requires surgery, for example, in stage 2 colorectal cancer treatment, chemotherapy may not be added because the risk of recurrence is low. However, some cases then recur. In addition, 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. In the treatment, 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. In addition, patients who have been classified into the same group and who received the same treatment may or may not relapse.

Also, in colorectal cancer treatment, examinations and examinations (follow-up) are performed at regular intervals in order to respond quickly to recurrence after surgery, but there is no good standard for predicting the presence or absence of recurrence, so let's reduce oversight Then, the number of follow-ups is increasing, and the current situation is that the insurance system covered by patients, medical institutions, and government funds is a burden.

In addition, it has 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.

On the other hand, in recent years, gene expression analysis (expression) analysis), in which genes expressed in cells in a certain state are comprehensively analyzed by comparing gene expression states, and their types and expression levels are compared between cells. Techniques have been developed for this purpose. For example, RNA-seq (Non-Patent Document 1) and CAGE (Cap Analysis Gene Expression: Non-Patent Document 2) that comprehensively analyze the expression state of the gene at the transcription start site as sequence information are known. Among these, 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.

However, the relationship between the expression level of the transcription initiation region in the human genome and a specific disease has never been reported so far.

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” .

That is, the present invention relates to the following 1) to 3).
1) For a biological sample derived from a cancer tissue separated from a colorectal cancer patient, the large intestine of the patient includes a step of measuring the expression level of one or more expression products of DNA including a transcription initiation region. To assess the risk of cancer metastasis or recurrence.
2) A test for evaluating the risk of metastasis or recurrence of colorectal cancer used in the method 1), which comprises an oligonucleotide that specifically hybridizes with the transcription product of DNA or an antibody that recognizes the translation product of DNA. For 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.

According to the present invention, for example, the presence or absence of recurrence risk can be determined quickly and accurately even in a patient without lymph node metastasis. In addition, since 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). In addition, by using the method of the present invention, a new classification method that can select an optimal treatment method with small variation in treatment effect can be established.

(A) Progression classification according to the 8th edition of the intestinal cancer handling agreement, (B) Progression classification according to UICC 7th edition Colorectal cancer handling rules 8th edition and TNM classification comparison table

In the present invention, colorectal cancer means a carcinoma that occurs in the large intestine (cecum, colon, rectum).

In the present invention, metastasis means that colon cancer grows in lymph nodes and other organs, and evaluation of metastasis means the presence or absence of metastasis or metastasis ability (colorectal cancer metastasizes to multiple organs and grows). To evaluate or measure the presence or absence of Preferable examples include evaluating or measuring liver metastasis or lymph node metastasis.

In the present invention, “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 For example, an equivalent stage in the progress classification (UICC 7th edition) by the TNM classification by the International Union for Cancer (UICC) can be mentioned. For reference, 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, and FIG. 2 shows a comparison table between the 8th edition of the Colorectal Cancer Handling Regulations and the TNM classification.

Stage 2 according to the 8th edition of the colorectal cancer handling protocol 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. In 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.

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.

In the present specification, the term “nucleic acid” or “polynucleotide” means DNA or RNA. In addition, “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.

In the present invention, a transcription product of DNA consisting of the nucleotide sequence shown in SEQ ID NOs: 1 to 443 (human genomic DNA consisting of a transcription initiation region and 100 bases continuous downstream thereof) is 1) in stage 2 as shown in the Examples In patients with colorectal cancer (male / female) who have not undergone postoperative chemotherapy, 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) Patients with colorectal cancer (male / female), specimens without liver metastasis and specimens with liver metastasis 5) Lymph node metastasis in patients with colorectal cancer (male / female) Specimens and lymph without As a result of comprehensive analysis of the expression state of DNA more than 100 bases downstream including the transcription start region on the genome, using the CAGE (Cap Analysis Analysis) analysis method for samples with metastasis, between the two groups. A significant difference was observed in the expression level (transcription activity). Specifically, the 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.

Therefore, 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.

1f) A biomarker for evaluating the risk of recurrence in a colorectal cancer patient (female) who is in the expression product stage 2 of DNA containing the transcription start site in SEQ ID NOS: 1 to 23 and has not undergone postoperative chemotherapy.
The DNA expression product is a marker that increases the expression level when the risk of recurrence is high.
1m) A biomarker for evaluating the risk of recurrence in a colorectal cancer patient (male) who is an expression product stage 2 of DNA containing the transcription start point in SEQ ID NOs: 24-37 and has not undergone postoperative chemotherapy.
The differential expression of the DNA is a marker that increases the expression level when the risk of recurrence is high.
2f) 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.
Among these, 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, and 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.
2m) A biomarker for assessing the risk of recurrence in a colorectal cancer patient (male) who has not undergone postoperative chemotherapy at the expression product stage 3a or 3b of the DNA containing the transcription start site in SEQ ID NOs : 64-179.
The DNA expression product is a marker that increases the expression level when the risk of recurrence is high.
3f) A biomarker for evaluating the risk of recurrence in colorectal cancer patients (female) who have undergone postoperative chemotherapy at the expression product stage 3a or 3b of the DNA containing the transcription start site in SEQ ID NOs: 180 to 217 .
The DNA expression product is a marker that increases the expression level when the risk of recurrence is high.
3m) 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.
4m) 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).
Among these, 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, and 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.
5f) DNA expression product containing the transcription start site in SEQ ID NOs: 289 to 299 Biomarker for evaluating lymph node metastasis in colorectal cancer patients (female).
The expression product of the DNA is a marker that increases the expression level when the possibility of lymph node metastasis is high.
5m) 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).
Among these, 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, and 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.

In the present invention, 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. In 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 In other words, 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. In the present specification, such a transcription start region is also referred to as “a transcription start region shown in SEQ ID NOs: 1 to 443”.

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.

In the present invention, 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.

Here, the number of bases in the downstream base sequence may be any number that can identify the expression product. Examples of 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. Moreover, as the said base number, 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. In addition, if 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. . Here, the substantially identical base sequence is, for example, using the homology calculation algorithm NCBI BLAST, expectation value = 10; allow gap; filtering = ON; match score = 1; mismatch score = −3 When searching, it means that there is 90% or more, preferably 95% or more, and still more preferably 98% or more identity with the base sequence shown in SEQ ID NOs: 1 to 443.

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.
Among these, when 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.

2f) Transcription in SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 57, or SEQ ID NO: 56 in the assessment of recurrence risk in colorectal cancer patients (women) who are stage 3a or 3b and have not undergone postoperative chemotherapy An expression product of DNA containing the starting point.
2m) Sequence number 134, SEQ ID NO: 111, SEQ ID NO: 65, SEQ ID NO: 94, sequence in the evaluation of recurrence risk of colorectal cancer patients (male) who are stage 3a or 3b and have not undergone postoperative chemotherapy An expression product of DNA comprising the transcription start site in SEQ ID NO: 137 or SEQ ID NO: 177.
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).
In the evaluation of lymph node metastasis of 5m) colorectal cancer patient (male),
SEQ ID NO: 330, SEQ ID NO: 348, SEQ ID NO: 422, SEQ ID NO: 406, SEQ ID NO: 353, SEQ ID NO: 365, SEQ ID NO: 339, SEQ ID NO: 395, SEQ ID NO: 372, SEQ ID NO: 311, SEQ ID NO: 368, SEQ ID NO: 389, SEQ ID NO: 350, SEQ ID NO: 356, SEQ ID NO: 383, SEQ ID NO: 349, SEQ ID NO: 363, SEQ ID NO: 344, SEQ ID NO: 433, SEQ ID NO: 408, SEQ ID NO: 371, SEQ ID NO: 307, SEQ ID NO: 357, SEQ ID NO: 370, SEQ ID NO: 327, SEQ ID NO: 418, SEQ ID NO: 434, SEQ ID NO: 385, SEQ ID NO: 340, SEQ ID NO: 390, SEQ ID NO: 440, SEQ ID NO: 430, SEQ ID NO: 325, SEQ ID NO: 328, SEQ ID NO: 375, SEQ ID NO: 423, SEQ ID NO: 407, SEQ ID NO: 305, SEQ ID NO: 333, SEQ ID NO: 411, SEQ ID NO: 362, SEQ ID NO: 3 3, SEQ ID NO: 364, SEQ ID NO: 399, SEQ ID NO: 424, SEQ ID NO: 354, SEQ ID NO: 441, SEQ ID NO: 382, SEQ ID NO: 379, SEQ ID NO: 361, SEQ ID NO: 323, SEQ ID NO: 304, SEQ ID NO: 431, SEQ ID NO: 404, Sequence number 426, sequence number 415, sequence number 377, sequence number 360, sequence number 435, sequence number 419, sequence number 337, sequence number 367, sequence number 400, sequence number 405, sequence number 391, sequence number 378, sequence number 335, SEQ ID NO: 388, SEQ ID NO: 313, SEQ ID NO: 402, SEQ ID NO: 386, SEQ ID NO: 319, SEQ ID NO: 318, SEQ ID NO: 409, SEQ ID NO: 429, SEQ ID NO: 321, SEQ ID NO: 308, SEQ ID NO: 373, SEQ ID NO: 312, Sequence number 342, sequence number 381, sequence number 396, sequence number 416, sequence No. 343, SEQ ID NO: 351, SEQ ID NO: 421, SEQ ID NO: 413, SEQ ID NO: 366, SEQ ID NO: 324, SEQ ID NO: 394, SEQ ID NO: 401, SEQ ID NO: 437, SEQ ID NO: 380, SEQ ID NO: 439, SEQ ID NO: 412, SEQ ID NO: 398 , SEQ ID NO: 310, SEQ ID NO: 438, SEQ ID NO: 428, SEQ ID NO: 336, SEQ ID NO: 425, SEQ ID NO: 397, SEQ ID NO: 329 or SEQ ID NO: 417.

In addition, 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. Alternatively, 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.

In addition, for the purpose of further improving accuracy, these can be used in combination of two sets or three sets or more as appropriate. Further, 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. Here, as 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.

In addition, the expression level comprehensively means the expression level and activity of the expression product.

When 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.

Here, 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. This corresponds to the probes for detection in the above, and these can be designed based on the nucleotide sequences represented by SEQ ID NOs: 1 to 443. Here, “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. This means that 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.

Specifically, 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 can be used. Here, 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. In addition, “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 | sequence. The identity of the base sequence can be determined by the algorithm such as BLAST.

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.

Here, the “oligonucleotide” can be DNA or RNA, and can be synthesized or natural. Alternatively, the probe used for hybridization is usually labeled.

For example, when using the Northern blot hybridization method, 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.

When using the RT-PCR method, first, 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. Thereafter, PCR is performed according to a conventional method, and the obtained amplified double-stranded DNA is detected. For 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.

When measuring the expression level of mRNA in a sample using a DNA microarray, at least one nucleic acid (cDNA or DNA) derived from the expression product of the present invention (in this case, a transcription product) is immobilized on a support. By using 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. For example, the expression product of the present invention (transcription) The product may be a nucleic acid having the entire sequence or a partial sequence. Here, the “partial sequence” includes a nucleic acid consisting of at least 15 to 25 bases.

Here, 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%. As a more stringent hybridization condition, 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).

In addition, 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.

When measuring the expression level using the CAGE method, it can be carried out in accordance with the method described in Examples below.

In addition, a protein (ie, translation product) encoded by 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. In this case, 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.

For example, when a protein is used as a measurement target, an antibody against the expression product of the present invention (in this case, a translation product) is brought into contact with a biological sample, the polypeptide in the sample bound to the antibody is detected, and the level is detected. This is done by measuring. For example, according to Western blotting, after using the above-described antibody as a primary antibody, 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.

On the other hand, 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.

Thus, 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.

Here, the “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.

For example, 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.

Also, 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. In this case, as 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. And the expression level of the expression product in the patient-derived biological sample is compared with a standard value (for example, a range of ± 2SD is set as an allowable range). For example, when the expression level of the expression product in a patient-derived biological sample is higher (or lower) than the threshold level, the colorectal cancer of the patient can be evaluated as having no metastasis, low metastatic potential, or low recurrence risk. .

In accordance with the method of the present invention, 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. Specifically, a 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. And a reagent for immunological measurement including an antibody that recognizes). Oligonucleotides, antibodies and the like included in the kit can be obtained by known methods as described above.

In addition to the antibody and nucleic acid, the test kit can contain a labeling reagent, a buffer solution, a chromogenic substrate, a secondary antibody, a blocking agent, instruments and controls necessary for the test, and the like.

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>
-Eight samples for transcription start region extraction (no recurrence: 7 specimens, with recurrence: 1 specimen)
・ Verification sample 3 specimens (no recurrence: 2 specimens, recurrence: 1 specimen)
<3f: Colorectal cancer patients who have undergone postoperative chemotherapy who are women at advanced stage 3a or 3b>
・ Transcription start region extraction / 25 samples for evaluation (no recurrence: 17 specimens, recurrence: 8 specimens)
<3m: male with advanced stage 3a or 3b who received postoperative chemotherapy for colorectal cancer>
・ Transcription start region extraction / evaluation sample 38 specimens (no recurrence: 30 specimens, recurrence: 8 specimens)
<4f: Female colorectal cancer patients with or without liver metastases>
-Eight samples for transcription start region extraction (with liver metastasis: 3 samples, without liver metastasis: 5 samples)
・ Samples for verification 3 specimens (with liver metastasis: 1 specimen, without liver metastasis: 2 specimens)
<4m: Male colorectal cancer patients with or without liver metastases>
・ Nine samples for extracting transcription start region (with liver metastasis: 3 samples, without liver metastasis: 6 samples)
・ Samples for verification 3 specimens (with liver metastasis: 1 specimen, without liver metastasis: 2 specimens)
<5f: Female colorectal cancer patient with or without lymph node metastasis>
・ Sample 6 for extracting transcription start region (with lymph node metastasis: 2 samples, without lymph node metastasis: 4 samples)
・ Sample 3 for verification (with lymph node metastasis: 1 sample, without lymph node metastasis: 2 samples)
<5m: Male colorectal cancer patients with or without lymph node metastasis>
・ Eight samples for extracting transcription start region (with lymph node metastasis: 3 samples, without lymph node metastasis: 5 samples)
・ Sample 3 for verification (with lymph node metastasis: 1 sample, without lymph node metastasis: 2 samples)

(2) Storage and preparation of sample The extracted 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.

(3) Preparation of RNA The sample subjected to the crushing and extraction treatment 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. In addition, 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.

(4) CAGE library preparation 5 μg 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). Specifically, 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. After digesting and purifying the single-stranded RNA portion with RNase I, only the 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. After adapters necessary for sequencing were connected to both ends of the collected cDNA, 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.

(5) RNA expression analysis i) Preparation of reference transcription initiation region Project to profile the activity of transcription initiation sites measured in genome-wide for a total of about 1000 human samples including human primary cultured cells, cell lines, and tissues. Among the transcription initiation regions identified in “FANTOM5” (submitting paper), about 180,000 transcription initiation regions defined on the human reference genome hg19 were used as reference transcription initiation regions.

ii) Quantification of transcriptional activity The reads obtained by sequencing and the human reference genome (hg19) were aligned using bwa (Bioinformatics. 2009 Jul 15; 25 (14): 1754-60). Only alignments with a mapping quality of 20 or more and an alignment start position within the reference transcription start region were selected, and the number of reads for each transcription start region was counted. Using the total number of reads in each library and the library size estimated by the RLE (Genome Biol. 2010; 11 (10): R106) method, the count is converted to counts per million. .

(6) Results (6-1)
1f: Evaluation of recurrence risk of colorectal cancer patients (female) who have not undergone postoperative chemotherapy at stage 2 1m: patients of colorectal cancer patients (male) who have not received postoperative chemotherapy at stage 2 Evaluation of recurrence risk 3f: Evaluation of recurrence risk of colorectal cancer patients (women) who have undergone postoperative chemotherapy at stage 3a or 3b 3m: Large intestine undergoing postoperative chemotherapy at stage 3a or 3b Of risk of recurrence in cancer patients (male)

(A) Extraction of transcription start regions with different activities Regarding the transcriptional activity in the sample for transcription start region extraction / evaluation quantified above, from clinical specimen-derived profile group obtained from “no recurrence”, from “with recurrence” Difference analysis between the obtained clinical specimen-derived profile groups was performed using the R / Bioconductor edgeR package (Bioinformatics. 2010 Jan 1; 26 (1): 139-40). In other words, whether the average expression level is different between the two groups (if the average hypothesis is equal to the null hypothesis and this null hypothesis is true, the probability that the measurement result will occur by chance Is calculated statistically. When a 5% FDR (false discovery rate) was set as a threshold, DNAs containing transcription initiation regions smaller than this were identified (Tables 1-2, 3-1-3-2 and Tables 4-1-4), respectively. -2). This criterion is statistically estimated that 95% of the candidates extracted by the corresponding threshold are truly differential in expression, assuming that there is no differential expression normally used (P value) This is a stricter standard than when the probability of accidental occurrence is 5%.

(B) Classification of the presence or absence of recurrence using the transcription start regions shown in Tables 1 and 2, Tables 3-1 and 3-2, and Tables 4-1 and 4-2. Specimen groups a, b, and c), and the expression levels in Tables 1 and 2, Tables 3-1 and 3-2, and Tables 4-1 and 4-2 in 2/3 specimens (Sample groups b and c) A logistic regression model, which is a kind of generalized linear model, is constructed with the explanatory variables and their presence / absence as explanatory variables. By applying the expression levels in Tables 1 and 2, Tables 3-1 and 3-2, and Tables 4-1 and 4-2 in the remaining 1/3 (sample group a) specimens, Make a prediction. By performing the same prediction for the sample group b and the sample group c, predictions for all the samples were made from the sample group that did not include them.
When the results were compared with the presence or absence of recurrence, the following correct answer rates could be obtained respectively.
(1f) Assessment of recurrence risk in patients with advanced stage 2 colorectal cancer who have not undergone postoperative chemotherapy: Correct response rate: Approximately 90%
(1m) Assessment of recurrence risk in colorectal cancer patients who are advanced stage 2 men who have not undergone postoperative chemotherapy: Correct answer rate: Approximately 85%
(3f) Evaluation of recurrence risk of colorectal cancer patients who have undergone postoperative chemotherapy for women with advanced stage 3a or 3b: Correct response rate: Approximately 72%
(3m) Assessment of risk of recurrence in patients with advanced stage 3a or 3b who received postoperative chemotherapy for colorectal cancer: correct answer rate: about 90%

(6-2)
2f: Assessment of recurrence risk of colorectal cancer patients (female) who have not undergone postoperative chemotherapy at stage 3a or 3b 2m: Patients with colorectal cancer who have not undergone postoperative chemotherapy at stage 3a or 3b 4f: Evaluation of liver metastasis of colorectal cancer patient (female) 4m: Evaluation of liver metastasis of colorectal cancer patient (male) 5f: Evaluation of lymph node metastasis of colorectal cancer patient (female) Evaluation 5m: Evaluation of lymph node metastasis in colorectal cancer patients (male)

(A) Extraction of transcription start regions with different activities Regarding the transcriptional activity in each sample for transcription start region extraction / evaluation determined above, “with recurrence” and “without recurrence”, “with liver metastasis” and “liver” Difference analysis between each clinical specimen-derived profile group obtained from “no metastasis” or “with lymph node metastasis” and “no lymph node metastasis” was performed in the R / Bioconductor edgeR package (Bioinformatics. 2010 Jan 1; 26 ( 1): 139-40). In other words, whether the average expression level is different between the two groups (if the average hypothesis is equal to the null hypothesis and this null hypothesis is true, the probability that the measurement result will occur by chance Is calculated statistically. When FDR (false discovery rate) 5% was set as a threshold, DNAs containing transcription initiation regions smaller than this were identified (Table 5, Tables 6-1 to 6-7, Tables 7 to 9, and Table 10). 1-10-6). This criterion is statistically estimated that 95% of the candidates extracted by the corresponding threshold are truly differential in expression, and the commonly used P value (assuming there is no differential expression) This is a stricter standard than when the probability of accidental occurrence is 5%.

(B) Selection of transfer start region for high-precision prediction (1)
Consider whether it is possible to classify the presence or absence of metastasis or the presence or absence of recurrence using only one expression level among the transcription initiation regions identified in (A) above. By setting some threshold for each, it was confirmed that both the transcription start region extraction sample and the verification sample can be classified with 100% specificity and 100% sensitivity. Examples of the threshold values are shown below (when the maximum value in one group is smaller than the minimum value in the other group, the average of these values is shown in Tables 11 to 15). 2f) DNA for evaluating the risk of recurrence in patients with colorectal cancer (female) who have not undergone postoperative chemotherapy at stage 3a or 3b

2m) DNA for assessing recurrence risk in colorectal cancer patients (male) who have not undergone postoperative chemotherapy at stage 3a or 3b

4f) DNA for evaluation of liver metastasis of colorectal cancer patients (women)

4m) DNA for evaluation of liver metastasis in colorectal cancer patients (male)

5m) DNA for evaluation of lymph node metastasis in colorectal cancer patients (male)

(C) Selection of transfer start region for performing highly accurate prediction (2)
It is considered whether the presence or absence of metastasis or the presence or absence of recurrence can be classified using a plurality of the expression levels of the transcription initiation region identified above. As an example, consider the construction of a logistic regression model, which is a kind of generalized linear model. This is to probabilistically predict the dependent variable (Y) indicating the presence or absence of recurrence or lymph node metastasis with an explanatory variable (Xi, logarithm of the above counts per million) that is the expression level of the transcription initiation region. Is one of the simplest models.

For all two combinations selected from the above transcription start regions, using these expression levels as explanatory variables, construct a logistic regression model to estimate the presence or absence of recurrence or lymph node metastasis for the sample for transcription start region extraction, A sample that can be classified with 100% specificity and 100% sensitivity was selected for both the transcription start region extraction sample and the verification sample (Tables 16 to 20).

Here, the logistic regression model, which is one of the simplest of machine learning devices, is used, but the method of measuring the expression of the transcription start region, the expression level and genotype of other genes, etc. By using other mathematical models appropriately by combination or the like, more robust prediction is possible. 2f) DNA for evaluating the risk of recurrence in patients with colorectal cancer (female) who have not undergone postoperative chemotherapy at stage 3a or 3b

2m) DNA for assessing recurrence risk in colorectal cancer patients (male) who have not undergone postoperative chemotherapy at stage 3a or 3b

4m) DNA for evaluation of liver metastasis in colorectal cancer patients (male)

5f) DNA for evaluating lymph node metastasis of colorectal cancer patients (female)

5m) DNA for evaluation of lymph node metastasis in colorectal cancer patients (male)

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. As a result, 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).

According to 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.

Claims (31)

1. A step of measuring the expression level of one or more expression products of DNA containing a transcription initiation region for a biological sample derived from a cancer tissue isolated from a colorectal cancer patient; A method to assess the risk of metastasis or recurrence.
2. The DNA is a DNA comprising a base at an arbitrary position of the transcription start region in the base sequence represented by SEQ ID NOs: 1 to 443 and at least one base continuous downstream thereof, and the transcription start region is represented by SEQ ID NOs: 1 to The method according to claim 1, wherein both ends are defined by the first base of the base sequence represented by 443 and the 101st base from the 3 ′ end.
3. The DNA is a DNA comprising a base at an arbitrary position of the transcription start region in the base sequence represented by SEQ ID NOs: 1 to 253 and at least one base continuous downstream thereof, and the transcription start region is represented by SEQ ID NOs: 1 to The method according to claim 1 or 2, wherein the recurrence risk of colorectal cancer, which is a region defined at both ends by the first base of the base sequence represented by 253 and the 101st base from the 3 'end, is evaluated.
4. The DNA is a DNA comprising a base at an arbitrary position of a transcription initiation region in the base sequence represented by SEQ ID NOs: 254 to 288 and at least one base continuous downstream thereof, and the transcription initiation region comprises SEQ ID NOs: 254 to The method according to claim 1 or 2, wherein the liver metastasis of colorectal cancer, which is a region defined at both ends by the first base of the base sequence represented by 288 and the 101st base from the 3 'end, is evaluated.
5. The DNA is a DNA comprising a base at an arbitrary position in the transcription initiation region in the base sequence represented by SEQ ID NOs: 289 to 443 and at least one base continuous downstream thereof, and the transcription initiation region is represented by SEQ ID NOs: 289 to 289 The method according to claim 1 or 2, wherein lymph node metastasis of colorectal cancer, which is a region defined at both ends by the first base of the base sequence represented by 443 and the 101st base from the 3 'end, is evaluated.
6. The DNA is a DNA comprising a base at an arbitrary position of the transcription start region in the base sequence represented by SEQ ID NOs: 1 to 23 and at least one base continuous downstream thereof, and the transcription start region is represented by SEQ ID NOs: 1 to A woman with colorectal cancer who has advanced stage 2 and is not undergoing postoperative chemotherapy, which is a region defined at both ends by the first base of the base sequence shown by 23 and the 101st base from the 3 ′ end The method according to claim 3, wherein the risk of recurrence of colorectal cancer in is evaluated.
7. The DNA is a DNA consisting of a base at an arbitrary position in the transcription initiation region in the base sequence represented by SEQ ID NOs: 24-37 and at least one base continuous downstream thereof, the transcription initiation region comprising: A male colorectal cancer patient who has advanced stage 2 and is not undergoing postoperative chemotherapy, which is a region defined at both ends by the first base of the base sequence shown by 37 and the 101st base from the 3 ′ end The method according to claim 3, wherein the risk of recurrence of colorectal cancer in is evaluated.
8. The DNA is a DNA comprising a base at an arbitrary position of the transcription start region in the base sequence represented by SEQ ID NOs: 38 to 63 and at least one base continuous downstream thereof, and the transcription start region is represented by SEQ ID NOs: 38 to 38 A female large intestine in which the end stage is defined by the first base of the base sequence represented by 63 and the 101st base from the 3 ′ end and which is not advanced postoperative chemotherapy in the stage 3a or 3b. The method according to claim 3, wherein the risk of recurrence of colorectal cancer in cancer patients is evaluated.
9. The DNA is a DNA comprising a base at an arbitrary position of the transcription start region in the base sequence represented by SEQ ID NOs: 64-179 and at least one base continuous downstream thereof, and the transcription start region is represented by SEQ ID NO: 64- The male large intestine in which the end stage is defined by the first base of the base sequence represented by 179 and the 101st base from the 3 ′ end and which is in the stage of progression stage 3a or 3b and not undergoing postoperative chemotherapy The method according to claim 3, wherein the risk of recurrence of colorectal cancer in cancer patients is evaluated.
10. The DNA is a DNA comprising a base at an arbitrary position in the transcription start region in the base sequence represented by SEQ ID NOs: 180 to 217 and at least one base continuous downstream thereof, and the transcription start region is represented by SEQ ID NOs: 180 to 180 Female colorectal cancer that has undergone postoperative chemotherapy in advanced stage 3a or 3b, which is a region defined at both ends by the first base of the base sequence represented by 217 and the 101st base from the 3 ′ end The method according to claim 3, wherein the risk of recurrence of colorectal cancer in a patient is evaluated.
11. The DNA is a DNA comprising a base at an arbitrary position in the transcription start region in the base sequence represented by SEQ ID NOs: 218 to 253 and at least one base continuous downstream thereof, and the transcription start region comprises SEQ ID NOs: 218 to Male colorectal cancer that has undergone postoperative chemotherapy in advanced stage 3a or 3b, which is a region defined at both ends by the first base of the base sequence represented by 253 and the 101st base from the 3 ′ end The method according to claim 3, wherein the risk of recurrence of colorectal cancer in a patient is evaluated.
12. The DNA is a DNA comprising a base at an arbitrary position of the transcription start region in the base sequence represented by SEQ ID NOs: 254 to 262 and at least one base continuous downstream thereof, and the transcription start region is represented by SEQ ID NOs: 254 to The liver metastasis of colorectal cancer in a female colorectal cancer patient, which is a region defined at both ends by the first base of the base sequence represented by 262 and the 101st base from the 3 'end, is evaluated. the method of.
13. The DNA is a DNA comprising a base at an arbitrary position in the transcription start region in the base sequence represented by SEQ ID NOs: 263 to 288 and at least one base continuous downstream thereof, and the transcription start region is represented by SEQ ID NO: 263 to The liver metastasis of colorectal cancer in a male colorectal cancer patient, which is a region defined at both ends by the first base of the base sequence represented by 288 and the 101st base from the 3 'end, is evaluated. the method of.
14. The DNA is a DNA comprising a base at an arbitrary position of a transcription initiation region in the base sequence represented by SEQ ID NOs: 289 to 299 and at least one base continuous downstream thereof, and the transcription initiation region comprises SEQ ID NOs: 289 to 289 The lymph node metastasis of colorectal cancer in a female colorectal cancer patient, which is a region defined at both ends by the first base of the base sequence represented by 299 and the 101st base from the 3 ′ end, is evaluated. The method described.
15. The DNA is a DNA comprising a base at an arbitrary position of the transcription start region in the base sequence represented by SEQ ID NOs: 300 to 443 and at least one base continuous downstream thereof, and the transcription start region is represented by SEQ ID NOs: 300 to 300 The lymph node metastasis of colorectal cancer in a male colorectal cancer patient, which is a region defined at both ends by the first base of the base sequence represented by 443 and the 101st base from the 3 ′ end, is evaluated. The method described.
16. The method according to claim 8, wherein the base sequences represented by SEQ ID NOs: 38 to 63 are the base sequences represented by SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 57, and SEQ ID NO: 56.
17. The method according to claim 9, wherein the base sequences represented by SEQ ID NOs: 64 to 179 are base sequences represented by SEQ ID NO: 134, SEQ ID NO: 111, SEQ ID NO: 65, SEQ ID NO: 94, SEQ ID NO: 137, and SEQ ID NO: 177.
18. The method according to claim 12, wherein the base sequences represented by SEQ ID NOs: 254 to 262 are the base sequences represented by SEQ ID NO: 261, SEQ ID NO: 258, SEQ ID NO: 262, and SEQ ID NO: 260.
19. The method according to claim 13, wherein the base sequence represented by SEQ ID NO: 263 to 288 is the base sequence represented by SEQ ID NO: 283, SEQ ID NO: 284 or SEQ ID NO: 278.
20. The nucleotide sequences represented by SEQ ID NOs: 300 to 443 are SEQ ID NO: 330, SEQ ID NO: 348, SEQ ID NO: 422, SEQ ID NO: 406, SEQ ID NO: 353, SEQ ID NO: 365, SEQ ID NO: 339, SEQ ID NO: 395, SEQ ID NO: 372, SEQ ID NO: 311, SEQ ID NO: 368, SEQ ID NO: 389, SEQ ID NO: 350, SEQ ID NO: 356, SEQ ID NO: 383, SEQ ID NO: 349, SEQ ID NO: 363, SEQ ID NO: 344, SEQ ID NO: 433, SEQ ID NO: 408, SEQ ID NO: 371, SEQ ID NO: 307, Sequence number 357, sequence number 370, sequence number 327, sequence number 418, sequence number 434, sequence number 385, sequence number 340, sequence number 390, sequence number 440, sequence number 430, sequence number 325, sequence number 328, sequence number 375, SEQ ID NO: 423, SEQ ID NO: 407, SEQ ID NO: 305, SEQ ID NO: 33 , SEQ ID NO: 411, SEQ ID NO: 362, SEQ ID NO: 393, SEQ ID NO: 364, SEQ ID NO: 399, SEQ ID NO: 424, SEQ ID NO: 354, SEQ ID NO: 441, SEQ ID NO: 382, SEQ ID NO: 379, SEQ ID NO: 361, SEQ ID NO: 323, SEQ ID NO: 323 SEQ ID NO: 304, SEQ ID NO: 431, SEQ ID NO: 404, SEQ ID NO: 426, SEQ ID NO: 415, SEQ ID NO: 377, SEQ ID NO: 360, SEQ ID NO: 435, SEQ ID NO: 419, SEQ ID NO: 337, SEQ ID NO: 367, SEQ ID NO: 400, SEQ ID NO: 405 , SEQ ID NO: 391, SEQ ID NO: 378, SEQ ID NO: 335, SEQ ID NO: 388, SEQ ID NO: 313, SEQ ID NO: 402, SEQ ID NO: 386, SEQ ID NO: 319, SEQ ID NO: 318, SEQ ID NO: 409, SEQ ID NO: 429, SEQ ID NO: 321, SEQ ID NO: 321 No. 308, SEQ ID NO: 373, SEQ ID NO: 312, SEQ ID NO: 342, SEQ ID NO: 381, SEQ ID NO: 396, SEQ ID NO: 416, SEQ ID NO: 343, SEQ ID NO: 351, SEQ ID NO: 421, SEQ ID NO: 413, SEQ ID NO: 366, SEQ ID NO: 324, SEQ ID NO: 394, SEQ ID NO: 401, SEQ ID NO: 437, SEQ ID NO: 380, SEQ ID NO: 439, SEQ ID NO: 412, SEQ ID NO: 398, SEQ ID NO: 310, SEQ ID NO: 438, SEQ ID NO: 428, SEQ ID NO: 336, SEQ ID NO: 425, SEQ ID NO: 397, SEQ ID NO: 329 and SEQ ID NO: 417. The method of claim 15.
21. The method according to claim 8, wherein the base sequences represented by SEQ ID NOs: 38 to 63 are combinations of the base sequences represented by the sequence numbers 1) to 2) below.
    

    
22. The method according to claim 9, wherein the base sequences represented by SEQ ID NOs: 64 to 179 are combinations of base sequences represented by SEQ ID NOs: 1) to 14) below.
    

    
23. The method according to claim 13, wherein the base sequences represented by SEQ ID NOs: 263 to 288 are combinations of base sequences represented by SEQ ID NOs: 1) to 10) below.
    

    
24. The method according to claim 14, wherein the base sequences represented by SEQ ID NOs: 289 to 299 are combinations of the base sequences represented by SEQ ID NOs: 1) to 3) below.
    

    
25. The method according to claim 15, wherein the base sequences represented by SEQ ID NOs: 300 to 443 are combinations of base sequences represented by SEQ ID NOs: 1) to 53) below.
    

    
26. The method according to any one of claims 1 to 25, further comprising the step of comparing the expression level of the expression product of the DNA with a control level.
27. The method according to any one of claims 1 to 25, further comprising a step of comparing the expression level of the expression product of the DNA with a threshold level.
28. The method according to any one of claims 1 to 27, wherein the expression level of the expression product is measured by measuring the amount of the transcription product or the amount of the translation product.
29. The metastasis of colorectal cancer used in the method according to any one of claims 1 to 28, which comprises an oligonucleotide that specifically hybridizes to the DNA or a transcription product of DNA, or an antibody that recognizes the translation product of the DNA. Or a test kit to assess the risk of recurrence.
30. 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.
31. The DNA is a DNA comprising a base at an arbitrary position in the transcription initiation region in the base sequence represented by SEQ ID NOs: 1 to 443 and one or more bases continuous downstream thereof;
    The use according to claim 30, wherein the transcription initiation region is a region defined at both ends by the first base of the base sequence represented by SEQ ID NOs: 1 to 443 and the 101st base from the 3 'end.
    

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