JP5586164B2 - How to determine cancer risk in patients with ulcerative colitis - Google Patents

Description

  The present invention relates to a method for determining cancer risk in patients with ulcerative colitis.

  Ulcerative colitis is a relatively benign type of colitis that has occurred in a wide range of ages and has a high onset frequency. Prognosis is good in ulcerative proctitis, where inflammation and ulcers are confined only to the rectum, with few serious complications. On the other hand, in about 10-30%, ulcerative proctitis spreads throughout the large intestine. Colon cancer is said to occur in about 1 in 100 patients each year in end-stage ulcerative colitis, and when ulcerative colitis is widespread in the intestine, 10 out of 100 people will develop colon cancer. Has been. In addition, it is said that the rate of progression to colon cancer is high when the disease duration is 8 years or longer (Non-patent Document 1).

  For these reasons, it is clinically important to predict the risk of canceration in patients with ulcerative colitis, and an evaluation method for predicting the risk of canceration is required in diagnostic treatment.

  RUNX3 is one type of transcription factor. It is suggested that methylation at the upstream site or deletion of the heterozygote (Loss-of-Heterozygosity, LOH) is a risk factor for developing gastric cancer (Non-patent document 2), and colon cancer (Non-patent document). 3) There is a report that the same applies to pancreatic cancer (Non-patent Document 4). On the other hand, although the RUNX3 mRNA expression level of the inflammatory ulcerative colitis patient in the long-term afflicted ulcerative colitis patient is shown to be increased compared to the normal site, the relationship with the canceration risk has not been shown ( Non-patent document 5).

  On the other hand, there is a DNA copy number as an index of other diseases, and this is used as an index independent of the mRNA expression level. In fact, it is known that there is a correlation between the DNA copy number and the gene expression level only in 3.8% of genes, and there is no correlation in most genes (Non-patent Document 6). In the last few years, a method for comprehensive search using the array CGH method or the like has been developed for analyzing the DNA copy number. Furthermore, a relationship between DNA copy number variation (Copy Number Variation, hereinafter simply referred to as CNV) and disease is suggested (Non-patent document 7, Non-patent document 8). A method for predicting the prognosis of a patient is also disclosed (Patent Document 1).

  However, no association has been reported between the risk of carcinogenesis in patients with long-term ulcerative colitis and the DNA copy number.

International Publication WO2007086351 Pamphlet Merck Manual Medical Encyclopedia latest home version published 2004/06/14 Cell 109: 113, 2002 Cancer Cell 14: 226, 2008 Br J Cancer 98: 1690, 2008 Inflamm Bowel Dis E-Publication 2008 / Jul / 30 Cancer Res 62: 1134, 2002 Nature 444: 444-54, 2006 Science 305: 525-8, 2004

  An object of the present invention is to provide an objective method for predicting cancer risk by targeting clinical samples of ulcerative colitis patients in diagnosis of ulcerative colitis patients.

  The present inventors analyzed the large intestine mucosal tissue of patients with ulcerative colitis accumulated through long-term clinical surveillance (gene expression profiling, mutation profiling of DNA copy number), so that the DNA copy number mutation and cancer associated with this disease A method for determining the risk of canceration in ulcerative colitis patients has been invented. Therefore, the present invention provides a method for determining the canceration risk of ulcerative colitis patients, a kit and a primer set for determining the canceration risk of ulcerative colitis patients described in the following section.

  Item 1. A method for determining a cancer risk of an ulcerative colitis patient using as an index the DNA copy number of RUNX3 in genomic DNA collected from the ulcerative colitis patient.

  Item 2. The method according to Item 1, wherein when the DNA copy number of RUNX3 in the colonic mucosa tissue derived from the subject is 2, it is determined that the subject has a high risk of developing cancer, and when the copy number is greater than 2 A method to determine that the risk of canceration is low.

  Item 3. Item 3. The method according to Item 1 or 2, comprising a step of measuring the DNA copy number of RUNX3 in genomic DNA collected from a patient with ulcerative colitis.

  Item 4. Item 4. The method according to Item 3, wherein the DNA copy number of RUNX3 is measured by PCR method, FISH method, array CGH method, DNA microarray method or Southern hybridization method.

  Item 5. If the measured value of the DNA copy number of RUNX3 by the PCR method is less than or equal to the preset boundary value A within the numerical range of 2.4 to 2.6, the copy number is assumed to be 2 and the onset of canceration in the subject Item 5. The method according to Item 4, wherein it is determined that the risk is high, and if the measured value is greater than the boundary value A, the risk of canceration is determined to be low because the copy number is greater than 2.

  Item 6. Item 6. The method according to any one of Items 1 to 5, wherein the base sequence of RUNX3 is represented by SEQ ID NO: 1 or 2.

  Item 7. A kit for determining the risk of canceration of a patient with ulcerative colitis comprising a reagent for measuring the DNA copy number of RUNX3.

  Item 8. Item 8. The kit according to Item 7, wherein the reagent for measuring the DNA copy number of RUNX3 is a RUNX3-specific primer or a RUNX3-specific probe that amplifies all or part of RUNX3.

  Item 9. Item 8. The reagent for measuring the DNA copy number of RUNX3, comprising a pair of RUNX3 specific primers that amplify a nucleic acid fragment containing the 8070211 to 8070141 region of the sequence represented by Genbank accession number NT_004610 kit.

  Item 10. A primer set comprising a combination of an oligonucleotide comprising the DNA sequence represented by SEQ ID NO: 5 and an oligonucleotide comprising the DNA sequence represented by SEQ ID NO: 6.

  ADVANTAGE OF THE INVENTION According to this invention, it can utilize for predicting the cancer risk of an ulcerative colitis patient objectively from the DNA copy number of RUNX3 with high test sensitivity.

Colorectal mucosal epithelial tissue (LI), general colorectal cancer (SC), non-cancer ulcerative colitis colorectal mucosal epithelial tissue (UC), cancer tissue with ulcerative colitis (UC-Ca) and ulcerative cancer Figure 5 shows a boxplot of DNA copy number of RUNX3 in colitis mucosal epithelial tissue (UCC).

  Hereinafter, embodiments of the present invention will be described in more detail.

Method for determining cancer risk of ulcerative colitis patients The present invention determines the cancer risk of ulcerative colitis patients using the DNA copy number of RUNX3 in genomic DNA collected from ulcerative colitis patients as an index Provide a method.

  The ulcerative colitis patient as a target of the method of the present invention refers to a patient diagnosed with ulcerative colitis, and includes both patients in active phase and remission phase.

  Here, ulcerative colitis is usually diagnosed by diagnosing the course and history of symptoms, performing colon examination with an endoscope, and excluding infectious diseases. Preferably, Ministry of Health and Welfare Specified Disease Intractable Inflammatory Intestinal Disorder Research Group Ulcerative Colitis Diagnosis Standards Revision Plan (1997 Research Report) or Welfare Science Research Fund Subsidy Specified Disease Countermeasure Research Project Survey on Intractable Inflammatory Intestinal Disorder Diagnose according to a study on the definition of intractable cases of ulcerative colitis (2002 research report).

  In the present invention, a patient having a high risk of canceration indicates a patient who is highly likely to suffer from cancer in the future.

  In the present specification, “RUNX3” refers to a gene that is a kind of transcription factor and encodes a protein having a molecular weight of about 44 KDalton as described above. The gene sequence information can be found in the NCBIweb site (http://www.ncbi.nlm.nih.gov/) database (Genbank) with RefSeq ID: NM_00103168 (human, isoform 1), RefSeq ID: NM_004350 (human) ) Etc. In addition, the RUNX3 gene is encoded in the 251640888 to 25098589th region of chromosome 1 (access number NC_000001). That is, it is coded in the 8024579-8090078th area of the array registered as the access number NT_004610 in the database. The amino acid sequences are registered in the same database as NP_001026850 (human, isoform 1), NP_004311 (human, isoform 2), etc. (For human nucleic acid and amino acid sequences, see: Bae, SC, et. al., Oncogene.8: 809-814, 1993).

  Here, in the method of the present invention, the DNA copy number of RUNX3 in genomic DNA collected from a subject who is a patient with ulcerative colitis (hereinafter, the subject indicates a patient with ulcerative colitis in the present invention) is measured. This method includes not only a method comprising a step and a step of determining a cancer risk from the measured DNA copy number, but also a method of determining a cancer risk from information on RUNX3 DNA copy number of a subject whose genomic DNA sequence is known. The

  In addition, since the present invention uses the copy number of RUNX3 in genomic DNA as an index, the tissue from which genomic DNA of ulcerative colitis patients is collected is not particularly limited. For example, for example, colon mucosa tissue, fecal examination, intestinal fluid Etc., and colonic mucosa tissue is preferable. In order to measure the DNA copy number of RUNX3 in the large intestine mucosa tissue derived from a subject, a biopsy sample, an isolated organ, a paraffin-embedded tissue sample, intestinal fluid, stool, or a cultured cell or tissue obtained therefrom is used. Good.

  The normal gene copy number of RUNX3 in the genome is 2 in human somatic cells, ie the diploid genome. However, when the ulcerative colitis patient is a population, the present inventors have a normal value of 2 for the DNA copy number of RUNX3 in many patients who have developed colorectal cancer, and have not developed colorectal cancer. We found that for many patients, the DNA copy number of RUNX3 was significantly greater than normal (eg, copy number 3).

  Therefore, in one embodiment of the present invention, according to the method of the present invention, the DNA copy number of RUNX3 in the colonic mucosa tissue derived from the subject is used as an index. When the copy number is predicted to be high and the copy number is greater than 2, it can be determined that the risk of canceration is low.

  Here, the DNA copy number of RUNX3 can be measured by, for example, PCR method, FISH method, array CGH method, DNA microarray method, Southern hybridization method, but is not limited to these methods.

Polymerase Chain Reaction (PCR) method The method for measuring the DNA copy number of RUNX3 using the PCR method can be carried out, for example, by the following procedure, but is not particularly limited thereto, and PCR conditions and the like are well-known CNV measurement. It can be set appropriately according to the method.

  PCR is performed using cancer cell genomic DNA prepared by the phenol / chloroform method, centrifugal column method, magnetic bead method or the like as a template.

  About PCR, the method using ABI Fast SYBR Green master mix (Applied Biosystems Inc.) described in the following Example can be illustrated. That is, 95 ° C. for 20 seconds (1 cycle), 95 ° C. for 1 second, 60 ° C. for 20 seconds, and 40 cycles. However, these conditions can be appropriately changed as long as they can be reproduced. Cases where the invention is carried out under the changed conditions are also included in the scope of the present invention. The quantitative value of the target DNA can be determined based on the number of PCR cycles (Ct value) when the PCR product reaches an arbitrary threshold value. Furthermore, the copy number of a gene can be calculated by correcting it with a quantitative value using a reference sequence with almost no copy number change between cancer and normal cells.

  Therefore, the measurement value of the DNA copy number of RUNX3 obtained using the PCR method is usually not a natural number, but is obtained in such a way that a number after the decimal point such as 2.323. Therefore, when the PCR method is used in the method of the present invention, preferably, the predetermined value is a boundary value, and the copy number is 2 when the measured value of the DNA copy number of RUNX3 is not more than the boundary value. That is, it is determined that the subject has a high risk of developing cancer, and when the measured value is greater than the boundary value, the copy number is determined to be greater than 2, that is, the risk of canceration is low. Here, the boundary value can be appropriately set by a preliminary experiment or the like as an upper limit value of the PCR measurement value that can be expected to have a copy number of two. For example, multiple copy number measurements are obtained by PCR from samples from one or more subjects that are known or likely to have a copy number of 2, and based on their standard error A boundary value can be set. In the present invention, the boundary value is preferably 2.4 to 2.6.

  When the DNA copy number of RUNX3 is measured by the PCR method, a RUNX3-specific primer such as an oligonucleotide primer that can amplify all or part of RUNX3 may be used. This RUNX3-specific primer may be capable of hybridizing to a region having a base sequence not found in other genomic DNAs in the entire region of RUNX3.

  The size of the primer is preferably about 17 to 25 bases. There is a tendency that good results can be obtained when the Tm values of the primers are aligned between the upstream primer and the downstream primer and set to about 55 to 65 ° C. Choose a pair with less complementarity between the primers so that the two primers do not anneal. In particular, in order to prevent a decrease in amplification efficiency due to the formation of primer dimers, the 3 ′ ends of each primer are designed so as not to be complementary continuously for 3 bases or more. Usually, in order to avoid the formation of secondary structure in the primer, a self-complementary sequence of 4 bases or more is not included. Usually, the GC content is about 40 to 60% so that it does not partially become GC or AT-rich. Further, usually, the primer is set so that the 3 'end of the primer and the template DNA are stably bound, and in particular, the 3' side of the primer is not AT-rich or GC-rich.

  The primer is preferably a sequence capable of specifically measuring the DNA copy number of RUNX3, more preferably a sequence of a neighboring region including a region encoding the RUNX3 gene on the genome, and homologous to the sequence of RUNX3 on the genome More preferably, a sequence capable of detecting the intron portion of RUNX3 on the genome, more preferably a sequence straddling the intron portion and exon portion of RUNX3 on the genome, and access number NT_004610 of the database (Genbank). A pair of RUNX3-specific primers that amplify a nucleic acid fragment containing the region 8070211 to 8070141 of the sequence represented by .18 is more preferred, and a primer set consisting of oligonucleotides represented by the sequences of SEQ ID NO: 5 and SEQ ID NO: 6 is particularly preferred Like .

  An example of the primer base sequence used in the PCR method is described below.

Forward primer: 5′-CCAACCACCTGCCCTCTATTCC-3 ′ (SEQ ID NO: 5)
Reverse primer: 5′-TTGGGTGAACACAGTGGATGGTCA-3 ′ (SEQ ID NO: 6)
As the reference genome, for example, LINE-1 (a large number of sequences present on the genome that is considered to have almost no change in the DNA copy number, whether cancer or normal) can be used, and an example of the primer base sequence Described below.

Forward primer: 5'-AAAGCCGCTCCAACTACATGG-3 '(SEQ ID NO: 7)
Reverse primer: 5′-TGCTTTGAATGCGTCCCAGAG-3 ′ (SEQ ID NO: 8)
Fluorescence in situ hybridization (FISH) method When the DNA copy number of RUNX3 is measured by the FISH method, the procedure is as follows, but it is not limited to these methods. The probe is preferably an oligo or polynucleotide probe capable of specifically hybridizing with RUNX3 (hereinafter, an oligo or polynucleotide probe capable of specifically hybridizing with RUNX3 is also referred to as a RUNX3 specific probe. is there). This RUNX3-specific probe should be capable of hybridizing to a region having a base sequence not found in other genomic DNA that can be present in cancer cells in the entire region of RUNX3.

  As a probe used in the FISH method, a DNA fragment having a target sequence, a PCR product, cDNA, a PAC clone, a BAC clone, or the like can be used. The in situ hybridization method has been developed as a method for confirming the presence and distribution of specific DNA or RNA (nucleic acid) in cells or tissues. The principle is that the probe nucleic acid having a base sequence complementary to a specific nucleic acid in the cell uses the property of specifically forming a complex (hybridization). This is because when the antigenic substance (hapten) or the like is labeled, the hybridized portion can be identified. As a conventional probe label, not only RI but also a fluorescent labeling method using a non-radioactive hapten such as biotin and digoxigenin may be used (reference: separate laboratory medicine, in situ identification method of protein / nucleic acid molecule) Toyama).

  The FISH method can be carried out according to a known method, and the procedure is exemplified below. The chromosome specimen may be a slide glass smear of isolated cancer cultured cells, or a slide specimen sliced from a tissue block containing cancer fixed in formalin and embedded in paraffin. Chromosome specimen slides are hardened (fixed) and prevented from falling out of the slide glass during the hybridization process, and then denatured by formamide treatment. In the FISH method using biotin as a label, biotin-dUTP (or dATP) is used to label the probe DNA, the DNA is thermally denatured, and then hybridized with single-stranded DNA. The formed biotin-labeled DNA and double-stranded DNA of genomic DNA are washed with a washing solution mainly composed of SSC buffer and treated with an avidin-FITC solution having high affinity for biotin. Furthermore, it wash | cleans by a SSC buffer series, an anti-fading agent is dripped, a cover glass is covered, It observes with a fluorescence microscope, and photograph is taken. Since the fluorescence spot in the cell indicates RUNX3, the number thereof is measured and the DNA copy number is measured.

Array Comparative Genomic Hybridization (Array CGH) method CGH method is a kind of FISH method that uses fluorescent dyes to identify which chromosomes are abnormal, but the conventional method has low resolution. However, it was difficult to identify the target gene from the obtained data of genomic abnormality. Since the genomic region which is the target for detecting an abnormal DNA copy number has been clarified this time, an abnormal DNA copy number can be detected by the following CGH method.

  The array CGH method may be performed by the following procedure, but is not limited to these methods. DNA is extracted from the subject-derived cells and normal cells used as controls, the subject-derived cell DNA is labeled with a green fluorescent dye (FITC), and the normal cell-derived DNA is labeled with a red fluorescent dye (Texas red). A mixed solution consisting of equal amounts of both labeled DNAs is prepared and hybridized. In the conventional method, blood collected from normal humans was cultured as a specimen to be hybridized, then cell division was stopped at the middle stage of mitosis, and it was used as a chromosome specimen slide smeared on a glass slide with the cell membrane exposed. By performing CGH using a slide glass in which the cloned DNA fragments were arrayed (array CGH), the DNA fragments were arrayed based on the fluorescence signal intensity ratio derived from labeled cancer and normal cell-derived DNA. The DNA copy number of cancer in the corresponding region can be quantified. As DNA to be arrayed, a BAC clone obtained by cloning a 100-kb human genome fragment, or a product obtained by increasing the number of cloned genomic fragments by using the BAC as a template by the DOP-PCR method can be used. (Reference: Array CGH diagnostic utilization guidebook, issued February 2008).

Similar to the DNA microarray method array CGH method, it is possible to detect a change in the DNA copy number of a specific genome region using a commercially available DNA microarray for SNP detection (oligonucleotide array or cDNA microarray). The standard protocol of each commercially available array can be used for the DNA preparation method and labeling method. DNA copy number can be quantified according to its standard protocol.

Southern hybridization method The Southern hybridization method is a classic method for detecting a target DNA by utilizing complementarity of nucleic acids. That is, genomic DNA prepared from cancer and normal cells is treated with an appropriate restriction enzyme and then subjected to agarose gel electrophoresis and fractionated according to the size of the DNA. Next, after denaturation to single-stranded DNA by alkali denaturation, it is transferred and immobilized on a filter such as nitrocellulose. This filter can be reacted with a probe in which a DNA fragment containing a target sequence is labeled with a radioisotope or the like to detect a change in the DNA copy number of the target gene region.

Kit for Determining Cancer Risk of Ulcerative Colitis Patients In addition, the present invention provides a kit for determining cancer risk of ulcerative colitis patients, which contains a reagent for measuring the DNA copy number of RUNX3. provide.

  Examples of the reagent for measuring the copy number of RUNX3 include RUNX3-specific primers and RUNX3-specific probes that amplify all or part of RUNX3 described above. These RUNX3-specific primers and RUNX3-specific probes are as described above.

  The kit may further contain a set of other reagents necessary for measurement of the DNA copy number of RUNX3 by PCR method, FISH method, etc., buffer solution, instruction manual, and the like. In addition to the method of using the kit, the instruction manual may include criteria for predicting the risk of canceration.

Primer set Furthermore, the present invention provides a set of primers consisting of a combination of an oligonucleotide consisting of the DNA sequence of SEQ ID NO: 5 and an oligonucleotide consisting of the DNA sequence of SEQ ID NO: 6, but measuring the DNA copy number of RUNX3 Therefore, the sequence is not limited to this sequence, and the above-described RUNX3-specific primer can be used as appropriate. By using the primer of the present invention, the DNA copy number of RUNX3 in the colonic mucosa epithelium derived from the subject can be measured by the PCR method, and the presence or absence of the cancer risk of the subject can be predicted based on this measurement result. it can.

  The present invention can be performed on specimens of patients diagnosed with or suspected of having ulcerative colitis.

  EXAMPLES Hereinafter, although this invention is demonstrated in detail based on an Example, this invention is not limited to these Examples.

RUNX3 DNA copy number by extracting DNA and RNA from biopsy tissue specimens at the time of surveillance endoscopy for early detection of cancer using ulcerative colitis patients mucosal epithelial tissues or surgical excision specimens And its expression level was measured. The subject of the analysis is a patient whose consent for research cooperation was obtained in writing. Specimens collected at the time of surgery are part of the resected specimen, and if tissue is collected during endoscopy, the right colon (ascending colon, transverse colon), left colon (descending colon, sigmoid colon) and rectal It is a tissue collected from a large intestine mucosa of a tumor part or a non-tumor part with biopsy forceps. This study was conducted according to ethical principles based on the Declaration of Helsinki and ethical guidelines for clinical research.

Example 1. Measurement of DNA copy number of RUNX3 In order to determine the DNA copy number of RUNX3, primers having the following sequences were designed.

Forward primer: 5′-CCAACCACCTGCCCTCTATTCC-3 ′ (SEQ ID NO: 5)
Reverse primer: 5′-TTGGGTGAACACAGTGGATGGTCA-3 ′ (SEQ ID NO: 6)
Using LINE-1 (a large number of sequences present on the genome that is considered to have almost the same DNA copy number as cancer or normal) as a reference genome, primers having the following sequences were designed.

Forward primer: 5'-AAAGCCGCTCCAACTACATGG-3 '(SEQ ID NO: 7)
Reverse primer: 5′-TGCTTTGAATGCGTCCCAGAG-3 ′ (SEQ ID NO: 8)
Genomic DNA of ulcerative colitis colonic epithelial tissue was prepared to 2 μg / mL using QIAAMP DNA mini kit (Qiagen Co., Ltd.) according to the instruction manual, and used for quantification of DNA copy number.

  The PCR reaction was performed using an ABI Fast SYBR Green master mix with an ABI 7900 sequence detector under the following PCR conditions. PCR reaction conditions are 95 ° C. for 20 seconds (1 cycle), 95 ° C. for 1 second, 60 ° C. for 20 seconds, and 40 cycles.

  The quantitative value of the target DNA was calculated from a regression equation obtained by creating a calibration curve based on the number of PCR cycles (Ct value) when the PCR product reached an arbitrary threshold. Furthermore, the DNA copy number of RUNX3 was calculated by the following formula.

(T (RUNX3) / T (LINE-1)) / (C (RUNX3) / C (LINE-1)) x 2
(Here, T is the quantitative value of RUNX3 or LINE-1 in the DNA of the tumor to be obtained, and C is the quantitative value of RUNX3 or LINE-1 in the genomic DNA derived from human (male) normal tissue used as a control) .)
Results Colonic mucosal tissues of patients with ulcerative colitis [35 cases, breakdown is 18 cases of non-cancer complication cases (shown as UC in FIG. 1), 17 cases of cancer complication cases (shown as UCC in FIG. 1)] Cancer tissue excised from colon cancer patients without ulcerative colitis (shown as SC in FIG. 1: 16 cases) and peripheral mucosal epithelial tissue (shown as LI in FIG. 1: 21 cases), and further ulcerative The distribution of the DNA copy number of RUNX3 in colorectal cancer complicated with colitis (indicated as UC-Ca in FIG. 1: 13 cases) is shown in FIG.

  In patients with ulcerative colitis that did not develop colorectal cancer (UC: 18 cases), the mean and standard deviation were 2.96 ± 0.13, which was significantly higher than 2. On the other hand, in the mucosal tissues of ulcerative colitis patients (UCC: 17 cases) who developed colorectal cancer, the average value was 1.93 ± 0.18, indicating a copy number near 2. The DNA copy number of RUNX3 in the colon cancer tissue (UC-Ca) combined with ulcerative colitis patients was 1.89 ± 0.17, indicating a copy number in the vicinity of 2. In colon cancer (SC) and colon cancer peripheral mucosal epithelial tissue (LI) surrounding colon cancer in patients without ulcerative colitis, the DNA copy numbers of RUNX3 were 1.88 ± 0.16 and 2.00 ± 0.14, respectively. Showed a value of about 2. As a result of a round-robin test in consideration of multiplicity using the Tukey-Kramer HSD test (Trans NY Acad Sci 16:88, 1953), the UC RUNX3 DNA copy number was significantly higher than any other. . From these results, it can be seen that among ulcerative colitis patients, those whose RUNX3 DNA copy number is greater than 2 have a low risk of developing colon cancer, and those whose RUNX3 DNA copy number is 2 have a high risk of developing colon cancer. .

  As described above, both colon cancer patients without ulcerative colitis (SC, LI) and ulcerative colitis patients combined with colon cancer (UCC, UC-Ca), regardless of the site from which the sample was collected The number of copies showed a value near 2. Therefore, according to the method of this invention, it turns out that canceration risk can be determined with high sensitivity irrespective of the collection | collection site | part of a sample.

Example 2 Determination of the risk of canceration in patients with ulcerative colitis Next, the risk of canceration was determined for the colonic mucosa tissue of patients with ulcerative colitis.

Specifically, first, in order to set the range of the measured value in PCR in which the DNA copy number of RUNX3 is expected to be normal, the measured value in the mucosal tissue around the cancer is used as a cutoff value (boundary value). Based on the standard error, A ₁ = 2.42 (2 + 3SD) and A ₂ = 2.56 (2 + 4SD) were set. For ulcerative colitis patients, when the DNA copy number of RUNX3 was below the cut-off value, it was determined that the risk of cancer complication was high (Table 1).

As apparent from Table 1 above, when the cut-off value is A ₁ = 2.42, 15 cases of cancer complications (17 cases) are correctly determined to have a large cancer complication risk, and the cancer complication risk is erroneously determined. Only two cases are judged to be small. On the other hand, out of non-cancer complication cases (18 cases), 12 cases are correctly determined as low risk of cancer complication, and only 6 cases are erroneously determined as high risk of cancer complication.

  In general, misjudgments that occur in risk assessment methods include misjudgment in which patients who are actually high in cancer risk are erroneously judged to be low in cancer risk, and patients who are actually low in cancer risk are miscarried. There is a misjudgment that judges that the risk is high. Here, in surveillance for early detection of cancer, it is important to detect all patients at risk of canceration. Therefore, among the above misjudgments, patients who are actually at high risk of canceration are mistakenly diagnosed with cancer. It is preferable to reduce the number of misjudgments that are determined to be small.

  In the case of this method, the test sensitivity of complication cases of ulcerative colitis is 88%, and the risk of cancer complication can be determined with very high sensitivity.

Furthermore, when the cut-off value is A ₂ = 2.56, all 17 cases among the cancer complication cases (17 cases) are correctly determined as having a large cancer complication risk. Therefore, in this case, a complication example of ulcerative colitis could be determined with a test sensitivity of 100%.

  It was found that the DNA copy number of RUNX3 is associated with cancer risk in patients with ulcerative colitis. This makes it possible to detect the risk of canceration of the subject, and this prediction result can be used for surveillance treatment for early detection of cancer.

  SEQ ID NO: 1 shows the gene DNA sequence of human-derived RUNX3 (isoform 1).

  SEQ ID NO: 2 shows the gene DNA sequence of human-derived RUNX3 (isoform 2).

  SEQ ID NO: 3 shows the amino acid sequence of human-derived RUNX3 (isoform 1).

  SEQ ID NO: 4 shows the amino acid sequence of human-derived RUNX3 (isoform 2).

  SEQ ID NO: 5 shows the DNA sequence of the forward primer used to determine the DNA copy number of RUNX3.

  SEQ ID NO: 6 shows the DNA sequence of the reverse primer used to determine the DNA copy number of RUNX3.

  SEQ ID NO: 7 shows the DNA sequence of the forward primer used for determining the DNA copy number of the reference genome sequence LINE-1.

  SEQ ID NO: 8 shows the DNA sequence of the reverse primer used for determining the DNA copy number of the reference genome sequence LINE-1.

Claims (10)

A method for determining the risk of canceration of ulcerative colitis patients using the DNA copy number of RUNX3 in genomic DNA collected from patients with ulcerative colitis as an index, wherein the DNA copy number of RUNX3 in colonic mucosal tissue derived from a subject Is determined to have a high risk of developing cancer when the subject is 2, and determines that the risk of canceration is low when the copy number is greater than 2. The method according to claim 1, wherein the genomic DNA collected from a patient with ulcerative colitis is collected from the colonic mucosa in a non-tumor area. The method of Claim 1 or 2 including the process of measuring the DNA copy number of RUNX3 in the genomic DNA extract | collected from the ulcerative colitis patient. The method according to claim 3, wherein the DNA copy number of RUNX3 is measured by a PCR method, FISH method, array CGH method, DNA microarray method or Southern hybridization method. If the measured value of the DNA copy number of RUNX3 by the PCR method is less than or equal to the preset boundary value A within the numerical range of 2.4 to 2.6, the copy number is assumed to be 2 and the onset of canceration in the subject The method according to claim 4, wherein it is determined that the risk is high, and if the measured value is greater than the boundary value A, it is determined that the risk of canceration is low because the copy number is greater than 2. 5. The method according to any one of claims 1 to 5, wherein the base sequence of RUNX3 is represented by SEQ ID NO: 1 or 2. The kit for determining the canceration risk of the ulcerative colitis patient for using for the method as described in any one of Claims 1-5 containing the reagent for measuring the DNA copy number of RUNX3. The kit according to claim 7, wherein the reagent for measuring the DNA copy number of RUNX3 is a RUNX3-specific primer or a RUNX3-specific probe that amplifies all or part of RUNX3. The kit according to claim 7, comprising a pair of RUNX3-specific primers as a reagent for measuring the DNA copy number of RUNX3, which amplifies a nucleic acid fragment comprising the nucleotide sequence region shown in SEQ ID NO: 9 . A primer for use in the method according to any one of claims 1 to 5, comprising a combination of an oligonucleotide comprising the DNA sequence represented by SEQ ID NO: 5 and an oligonucleotide comprising the DNA sequence represented by SEQ ID NO: 6. set.

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