WO2010137671A1 - ガンのリンパ節転移またはそのリスクを判定する方法及びそのための迅速判定キット - Google Patents
ガンのリンパ節転移またはそのリスクを判定する方法及びそのための迅速判定キット Download PDFInfo
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- WO2010137671A1 WO2010137671A1 PCT/JP2010/059049 JP2010059049W WO2010137671A1 WO 2010137671 A1 WO2010137671 A1 WO 2010137671A1 JP 2010059049 W JP2010059049 W JP 2010059049W WO 2010137671 A1 WO2010137671 A1 WO 2010137671A1
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Definitions
- the present invention relates to a method for determining lymph node metastasis of cancer and a rapid determination kit used therefor.
- Cancer cells leave the primary lesion and metastasize throughout the body via blood vessels and lymphatic vessels. In cancer surgery, it is necessary to remove the lesion as reliably as possible. Therefore, it is required to accurately detect metastasis and perform appropriate treatment according to the degree of metastasis. For this reason, diagnosis of lymph node metastasis of cancer cells is extremely important for selection of appropriate treatment.
- Diagnosis of lymph node metastasis of cancer cells can be roughly divided into image diagnosis performed before treatment and pathological diagnosis performed after treatment (surgery).
- diagnostic imaging PET (computed tomography), PET (positron emission tomography) or PET-CT with an integrated PET and CT, EUS (endoscopic ultrasound) Is used for detection of cancer lymph node metastasis (examination of the presence or absence of lymph node metastasis, etc.), but it is difficult to detect minute lymph node metastasis by these image diagnosis It is effective only to a limited extent.
- Pathological diagnosis is a method of observing a specimen prepared from a number of extracted lymph node tissues under a microscope. It is a reliable diagnostic method with high accuracy.
- molecular diagnostic techniques using molecular markers are important in diagnosing lymph node metastasis of cancer cells, and several techniques are known.
- Many of such molecular diagnostic methods known in the art include a protein (target protein) that is not expressed in normal cells or has a low expression level, and is highly expressed in cancer cells (a target protein) or a nucleic acid contained in a gene encoding the target protein ( This is a method for detecting a target nucleic acid (generic name of DNA, mRNA, cDNA, etc.) as a molecular marker.
- target proteins contained in lymph node tissues excised and removed from living bodies can be detected by immunoassay, and target nucleic acids can be detected using the LAMP method (loop-mediated isothermal amplification method) or PCR (polymerase chain reaction) method. Amplification is performed, and the amplification product is detected by a known method to diagnose (determine) cancer cell metastasis.
- LAMP method loop-mediated isothermal amplification method
- PCR polymerase chain reaction
- Patent Document 1 Japanese Patent Laid-Open No. 2007-175021 describes PIGR, CLDN3, LGALS4, AGR2, TACSTD1, GPX2, RAI3, TSPAN1, CKB, ELF3, FXYD3, CDH1, REG4, GDF-15, CLDN4 , OLFM ⁇ ⁇ 4, CD9, CDH17, SELEBP, LCN2, TMPRSS4, CFTR, TM4SF3, ID1, CYP2S1, TFF3, EHF, FAT, KLF5, SLC9A3R2, HOXB9, ATP1B1, PCK1, FCGBP Has proposed a lymph node metastasis marker used to determine the presence or absence of lymph node metastasis of cancer cells derived from colorectal cancer, comprising mRNA of a gene encoding the gene or a fragment thereof.
- 037421 is the database access number (serial number) of NM_003404 (G1592), NM_002128 (G2645), NM_052868 (G3031), NM_005034 (G3177), NM_001540 (G3753), NM_005722 (G3826), and NM_015315 (G4370).
- NM_003404 serial number
- NM_002128 G2645
- NM_052868 G3031
- NM_005034 G3177
- NM_001540 G3753
- NM_005722 G3826
- NM_015315 G4370
- Non-Patent Document 1 Erlinger TP et al., JAMA 2004; 291; 585-590
- Non-patent document 2 Shimada H. et al., J. Surg. Oncol. 2003; 83; 248-252
- Non-patent document 3 Hashimoto K.
- Non-Patent Document 4 Miyata Y. et al., Urology 2001; 58; 161-164) and ovarian cancer
- Non-patent Literature5 Hefler LA et al., Clin. Cancer Res. 2008; 14 ; 710-714 as a risk factor and prognostic factor.
- Non-Patent Document 6 (Nozoe T. et al., Am. J. Surg. 1998; 176 (4): 335-8) shows that liver metastasis and lymph node metastasis of serum CRP levels in patients with colorectal cancer.
- Non-patent document 7 (Nozoe T. et al., Am. J. Surg. 2001; 182 (2), 197-201) describes esophageal cancer patients.
- lymph node metastasis is associated with a preoperative increase in serum CRP value
- Non-Patent Document 8 (Ines G. et al., World J. Gastroenterol. 2006; 12 ( 23), 3746-3750) describes that high serum CRP levels and lymph node metastasis are associated with esophageal cancer patients.
- Non-patent document 9 Carlson CS et al., Am. J. Hum. Gen. 2005; 77; 64-77
- Non-Patent Document 10 Szalai AJ et al., J. Mol. Med. 2005; 83; 440-447.
- CRP-717T> C gene polymorphism may be related to lymph node metastasis (Non-patent Document 11: Motoyama et al., ⁇ Journal of Japanese Society of Gastroenterological Surgery '' Vol. 41, No. 7, page 1169, (July 2008), CRP-717T> C gene polymorphisms were not used in the method of determining lymph node metastasis of cancer cells because of low accuracy and metastasis could not be determined with a significant difference.
- the SNP identification number rs1205 (CRP1846C> T (rs1205)) gene polymorphism is a single nucleotide mutation in the non-transcribed region of the CRP gene, while it has been reported to correlate with a decrease in serum CRP concentration. It is a type.
- the present invention provides the following determination method and determination kit.
- a method for determining lymph node metastasis of cancer or its risk by identifying a gene polymorphism of a human C-reactive protein gene [2] The method according to 1 above, wherein the lymph node metastasis of cancer or its risk is determined by identifying a gene polymorphism of SNP identification number rs1205. [3] The method according to 2 above, wherein when the gene polymorphism of the SNP identification number rs1205 is T / T type, the risk is determined to be high risk. [4] The method according to any one of 1 to 3 above, wherein the genetic polymorphism is identified by binding to RFLP or a corresponding complementary strand sequence.
- a nucleic acid for analyzing a base of SNP identification number rs1205 of a human C-reactive protein gene comprising a base of SNP identification number rs1205 of a human C-reactive protein gene, and SEQ ID NO: 1 and SEQ ID NO: 2
- the sample used for identifying the gene polymorphism of the human C-reactive protein gene is selected from the group consisting of whole blood, leukocytes, cancer primary lesions, lymphatic vessels, and lymph node tissues The method described.
- CRP Creactive protein
- cytokines interleukin, tumor necrosis factor, interferon, transforming growth factor, etc.
- the method of the present invention is independent of various cytokine concentrations, This alone can effectively predict and determine lymph node metastasis of cancer cells.
- the method for determining lymph node metastasis of cancer using the SNP identification number rs1205 (CRP1846C> T (rs1205)) gene polymorphism of the present invention is simpler than the conventional method, it has extremely high accuracy. That is, by using the SNP identification number rs1205 (CRP1846C> T (rs1205)) gene polymorphism, it was possible to make a determination with a significant difference.
- the most reliable and least invasive treatment method can be selected from options such as radiation therapy, and the clinical significance in determining the treatment strategy is extremely large.
- diagnosis before treatment must rely on image diagnosis, but at present, the accuracy is low.
- reliable diagnosis is made by pathological diagnosis, but this is a diagnosis after treatment (surgery). It is possible to simultaneously solve the problems of diagnosis of lymph node metastasis of cancer cells.
- the sample for detecting the polymorphism of the SNP identification number rs1205 (CRP1846C> T ⁇ (rs1205)) is a lymphoid tissue (lymph node or lymph vessel). Therefore, the burden on the patient is reduced and the sample preparation is facilitated, so that the burden on the examiner can be reduced.
- CRP C-reactive protein
- CRP C-reactive protein
- CRP C-reactive protein
- CRP C-reactive protein
- CRP is a kind of acute phase protein produced mainly by hepatocytes in response to inflammation, and its serum concentration has been conventionally used as a marker for various acute or chronic inflammatory diseases. Its name is derived from the serum protein (presence in the ⁇ globulin fraction) that shows a sedimentation reaction with the C polysaccharide of S. pneumoniae, and the blood concentration from 0.2 ⁇ g / mL due to infection, inflammation, tissue damage, etc. It increases dramatically from a few hundred times to a thousand times.
- CRP is a pentamer having a molecular weight of about 130,000 and consisting of 5 identical subunits, and its amino acid sequence is homologous to a portion of the serum C protein, complement C1.
- a genetic polymorphism is generally defined genetically as a specific base change (mutation) in one gene that is present at a frequency of 1% or more in the population.
- a gene polymorphism of SNP identification number rs1205 (in this specification, CRP1846C> T (rs1205) gene polymorphism) may be preferably used.
- the gene polymorphism of SNP identification number rs1205 is a polymorphism at the 2148th base (base indicated by r) on the base sequence of the CRP gene described in Table A above. Note that r means G (guanine) or A (adenine).
- the SNP identification number rs1205 is a polymorphism at the 422nd base (base indicated by Y) on the base sequence described in Table B above. Y means C (cytosine) or T (thymine). By identifying whether this base type is C / C (wild), C / T (hetero), or T / T (homo), the present invention can detect cancer lymph node metastasis or its risk. Is determined.
- the SNP identification number rs1205 is the SNP identification number registered in the SNP database (dbSNP) of NCBI (National Center for Biotechnology Information) in the United States. The SNP information registered in rs1205 is the NCBI site (http: //www.ncbi.nlm.nih.gov/projects/SNP/).
- the identification of the SNP identification number rs1205 (CRP1846C> T (rs1205)) gene polymorphism can be performed by various known methods capable of detecting the gene polymorphism, and is not particularly limited.
- PCR-RFLP restriction fragment length polymorphism
- PCR-SSCP single chain conformation polymorphism
- PCR-SSO specific sequence oligonucleotide
- PCR-SSO method ASO allele specific oligonucleotide hybridization method
- dot hybridization method or Taq-Man-PCR method
- any one of the above identification methods can be applied after the test DNA is previously amplified by a PCR method or a gene amplification method according to the PCR method.
- the DNA chip or microarray method Invader method, TaqMan-PCR method, MALDI-TOF / MS (matrix) method using primer extension method or RCA method are used. It is preferable to use it.
- a method suitable for the case where the amount of test DNA is small and a method suitable for identification for a large number of test DNA will be described by taking typical methods as examples.
- a suitable method is to prepare a DNA sample from the patient using methods well known to those skilled in the art, then cleave the prepared DNA sample with restriction enzymes, and then size the DNA fragment to its size. This is a method in which separation is performed in response, and then the size of the detected DNA fragment is compared with a control.
- a DNA sample is first prepared from a patient, and then DNA containing the CRP gene is amplified. Furthermore, the amplified DNA is cleaved with a restriction enzyme. The DNA fragments are then separated according to their size and the size of the detected DNA fragment is compared to a control.
- Examples of such a method include a method using a restriction enzyme fragment length polymorphism (RFLP), a PCR-RFLP method, and the like. That is, if there is a mutation at the restriction enzyme recognition site, or if there is a base insertion or deletion in the DNA fragment produced by the restriction enzyme treatment, the size of the fragment produced after the restriction enzyme treatment will change compared to the control. . By amplifying a portion containing this mutation by PCR and treating with each restriction enzyme, these mutations can be detected as a difference in mobility of bands after electrophoresis.
- RFLP restriction enzyme fragment length polymorphism
- RNA prepared from a patient can be converted into cDNA with reverse transcriptase, and this can be cleaved with a restriction enzyme and then subjected to Southern blotting. It is also possible to examine the difference in mobility after amplifying DNA containing the CRP gene by PCR using this cDNA as a template, cleaving it with a restriction enzyme.
- the primers used in the present invention include all those capable of amplifying DNA containing the CRP gene.
- the base length of the primer is preferably 10 bases or more, more preferably 15 bases or more.
- Each primer may be a single oligonucleotide or a mixture of a plurality of oligonucleotides.
- Examples of primers in PCR include Forward primer: 5'-CTT ATA GAC CTG GGC AGT-3 '(SEQ ID NO: 1), Reverse primer: 5'-GGA GTG AGA CAT CTT CTT G-3' (SEQ ID NO: 2) It is done.
- An example of a restriction enzyme is Bst4CI. Materials and conditions other than primers in PCR, application of restriction enzymes, electrophoresis, detection, etc. may be the same as conventional methods.
- the probe DNA used in the Southern blotting described above is not particularly limited as long as it can hybridize to the target nucleic acid.
- probe DNA that can hybridize to a target nucleic acid a nucleic acid for analyzing the base of SNP identification number rs1205 of the human CRP gene, comprising the base of SNP identification number rs1205 of the human CRP gene, and SEQ ID NO: 1
- CRP gene should be obtained from patients' blood, peripheral blood leukocytes, skin cells, mucosal cells and other cells, tissues such as liver, kidney, adrenal gland, brain, uterus, hair, etc. using known extraction and purification methods. Can do. Moreover, as long as it contains the base part analyzed in this invention, it can be used as a CRP gene in this invention irrespective of the distinction of full length DNA or partial DNA. In other words, a DNA fragment of any length can be used as long as it contains the base of SNP identification number rs1205.
- a method suitable for identifying a large number of test DNAs is to prepare a substrate on which a DNA containing a CRP gene prepared from a patient and a nucleotide probe (synonymous with the above-mentioned probe DNA) that hybridizes with the DNA are fixed, Next, after bringing the DNA into contact with the substrate, a PCR gene polymorphism is detected by detecting DNA (target nucleic acid) hybridized with the nucleotide probe immobilized on the substrate.
- a DNA sample containing a CRP gene from a patient can be prepared by a method well known to those skilled in the art, as described above.
- the preparation of the DNA sample as described above, it is extracted from the patient's blood, peripheral blood leukocytes, skin cells, mucosal cells and the like, liver, kidney, adrenal gland, brain, uterus and other tissues, hair, etc.
- DNA containing CRP gene is obtained by PCR using genomic (chromosomal) DNA as a template. It is also possible to prepare.
- the prepared DNA sample can be labeled for detection by a method well known to those skilled in the art, if necessary.
- the DNA chip method uses a method in which many types of probe DNA are aligned and immobilized on a substrate such as glass, and then labeled DNA is hybridized to detect the label (fluorescence, etc.) signal on the probe.
- a genetic polymorphism such as SNP is detected by differentially detecting a complete match and a single base mismatch by hybridization.
- the TaqMan PCR method is a method that uses a fluorescently labeled allele-specific oligo and a PCR reaction with Taq DNA polymerase.
- the Invader method is a special case in which two types of reporter probes specific to each allele of a genetic polymorphism such as SNP and one type of invader probe are hybridized to the template DNA, and the DNA structure is recognized and cleaved. This is a method that combines cleavage of DNA with an enzyme having a unique endonuclease activity.
- the SniPer method As a method using the primer extension reaction, for example, the SniPer method can be adopted.
- the SniPer method is based on a method called RCA (rolling circle amplification) method, which synthesizes complementary-strand DNA continuously while DNA polymerase moves on it using a circular single-stranded DNA as a template. It is something to do.
- genetic polymorphisms such as SNP can be determined by measuring the presence or absence of a color reaction that occurs when DNA amplification occurs.
- the MALDI-TOF / MS method is a method using a mass spectrometer, and is basically a method of genotyping SNPs using the difference in mass of different single bases. There are a method using PCR amplification and a method using multiplex.
- the sequencing method is a method of analyzing the frequency of genetic polymorphisms (especially SNPs) such as SNP by amplifying the region containing the genetic polymorphism by PCR and sequencing the DNA sequence using Dye Terminator etc. .
- the determination method of the present invention can be applied at various stages, but is particularly useful in determining a treatment strategy. For example, in patients with esophageal cancer with a depth of submucosal, it is difficult to determine lymph node metastasis by conventional methods. In contrast, in the present invention, lymph node metastasis or its risk can be determined with high accuracy. Therefore, unnecessary lymph node dissection or the like can be performed to reduce QOL, or cancer can be performed without performing necessary lymph node dissection. It is also possible to avoid neglecting progress.
- the type of gun to which the method of the present invention can be applied is not particularly limited, but can be applied to all solid guns.
- the primary site is applicable to cancer of the esophagus, lung, breast, head and neck, stomach, large intestine, biliary tract, pancreas, uterus, ovary, bladder, kidney, urothelium, and prostate.
- Rapid determination kit for lymph node metastasis or its risk The rapid determination kit for lymph node metastasis or its risk of the present invention can be prepared by a method well known to those skilled in the art.
- a kit form for example, various reagents necessary for detecting a CRP gene polymorphism using the primer of the present invention can be packaged in advance to form a kit.
- various oligonucleotides necessary as a primer or loop primer of the present invention four types of dNTPs (dATP, dCTP, dGTP, and dTTP) that serve as substrates for nucleic acid synthesis, and the above template dependency having strand displacement activity
- dNTPs dATP, dCTP, dGTP, and dTTP
- Reagents necessary for detection of an object are provided as a kit.
- a probe DNA that can hybridize with the target nucleic acid may be kitted as a constituent reagent.
- lymph node means “lymph node” and “lymph vessel”.
- determining lymph node metastasis refers to determining the presence / absence / absence of cancer cells in lymph nodes
- Determining the risk of node metastasis includes determining whether or not cancer cells may metastasize from a primary lesion to a lymph node when an individual suffers from cancer.
- Example 1 This example was performed on 113 patients (all Japanese) with thoracic esophageal squamous cell carcinoma. Of these, 38 patients had esophagectomy during one year from April 2007 after confirming esophageal cancer by pathological diagnosis. The remaining 75 patients were randomized from those who had undergone esophagectomy between 2000 and 2007 and who followed the course. Disease classification was performed according to the 6th edition of the classification of tumor lymph node metastasis (TNM) by the International Association for Cancer.
- TNM tumor lymph node metastasis
- CRP polymorphisms include CRP-717C> T (rs2794521), CRP1059G> C (rs1800947), CRP1444C> T (rs1130864) as tumor necrosis factors TNF- ⁇ -238G> A, TNF- ⁇ -308G> A, TNF- ⁇ -1031T> C, TNF- ⁇ 250G> A, INF- ⁇ 874A> T, TGF- ⁇ 1 29T> C, IL-1 ⁇ - 31C> T, IL-1 ⁇ -511C> T, IL-1 receptor antagonist, IL-2-330T> G, IL-4-590C> T, IL-6-634G> C, IL-6 receptor 48892A> Eighteen gene polymorphisms.
- PCR for target nucleic acid amplification was performed by denaturing the extracted DNA at 95 ° C for 15 minutes, followed by 35 cycles of reaction at 95 ° C for 30 seconds, 56 ° C for 30 seconds, and 72 ° C for 30 seconds. Heated at 5 ° C. for 5 minutes.
- Bst4CI was added to the PCR amplification product obtained by the above operation, incubated at 65 ° C.
- lymph node metastases Of the 113 esophageal cancer patients examined, 62 (55%) had lymph node metastases and 51 (45%) had no lymph node metastases. In patients with confirmed lymph node metastases, cancer penetration was significantly higher (P ⁇ 0.05) than patients without lymph node metastasis, but age, sex, and preoperative nutritional status There was no significant correlation between tumor markers, tumor site and tumor size, squamous cells and intramural metastasis and the presence or absence of lymph node metastasis. (See Table 1, “Clinical characteristics of patients with or without lymph node metastasis”)
- CRP- In 717T / C and C / C genotype patients 12 patients had lymph node metastasis, 16 patients did not have metastasis, and CRP1444C> T (rs1130864) gene polymorphism had CRP1444C / C genotype 56 patients had lymph node metastasis and 49 patients had no metastasis. (See Table 2, “CRP gene polymorphisms and lymph node metastasis”)
- lymph node metastasis was particularly difficult, and the ability to diagnose lymph node metastasis before treatment was limited to patients with submucosal esophageal cancer (33 cases) whose treatment method greatly affected, the diagnostic ability was extremely high.
- the sensitivity of lymph node metastasis diagnosis using state-of-the-art diagnostic imaging equipment is 50%, specificity is 79%, positive predictive value is 54%, negative predictive value is 68%
- diagnosis using the CRP1846C> T (rs1205) gene polymorphism showed better sensitivity with 64% sensitivity, specificity 79%, positive predictive value 69%, and negative predictive value 75% (Table 5 “ CRP1846C> T (rs1205) gene polymorphism or prediction of lymph node involvement in submucosal esophageal cancer when using conventional methods (CT and ultrasound tomography)).
- lymph node metastasis or its risk can be determined with high accuracy.
- CRP1846C> T rs1205 gene polymorphism
- Example 3 The relationship between CRP1846C> T (rs1205) gene polymorphism and lymph node metastasis was analyzed for 64 of 113 patients of Example 1 whose wall depth was pT1-2.
- the method according to the present invention can reliably determine lymph node metastasis even in earlier cancers, which may be overlooked by the conventional diagnostic imaging method, and is confirmed to be able to meet clinical requirements. It was done.
- Example 4 Among 152 patients of Example 2, 144 patients who had findings regarding lymphatic vessel invasion were analyzed for the relationship between CRP1846C> T (rs1205) gene polymorphism and lymphatic vessel invasion.
- “Lymph vessel invasion” refers to the condition where the presence of cancer cells is observed in the lymph vessels at the primary site, suggesting the possibility of future “lymph node metastasis” even if there is no “lymph node metastasis”. . Therefore, identification of the CRP1846C> T (rs1205) gene polymorphism is useful for risk assessment (earlier prediction) of “lymph node metastasis”.
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Abstract
Description
[1] ヒトC反応性タンパク質遺伝子の遺伝子多型を同定することにより、ガンのリンパ節転移またはそのリスクを判定する方法。
[2] SNP識別番号rs1205の遺伝子多型を同定することにより、ガンのリンパ節転移またはそのリスクを判定する前記1に記載の方法。
[3] SNP識別番号rs1205の遺伝子多型がT/Tタイプであるときに高リスクと判定する前記2に記載の方法。
[4] 遺伝子多型の同定をRFLPまたは対応する相補鎖配列との結合により行なう前記1~3のいずれかに記載の方法。
[5] 遺伝子多型の同定をPCR-RFLPにより行なう前記4に記載の方法。
[6] PCRにおけるプライマーとして Forwardプライマー:5'-CTT ATA GAC CTG GGC AGT-3'(配列番号1)、Reverseプライマー:5'-GGA GTG AGA CAT CTT CTT G-3' (配列番号2)を用い、制限酵素としてBst4CIを用いる前記5に記載の方法。
[7] ガンが固形ガンである前記1~6のいずれかに記載の方法。
[8] ヒトC反応性タンパク質遺伝子の塩基配列のSNP識別番号rs1205を含む領域を増幅するためのプライマーとRFLPによりSNP識別番号rs1205の遺伝子多型を判定するための制限酵素を含むガンのリンパ節転移またはそのリスクを判定するためのPCR-RFLP用迅速判定キット。
[9] プライマー対としてForwardプライマー:5'-CTT ATA GAC CTG GGC AGT-3' (配列番号1)、Reverseプライマー:5'-GGA GTG AGA CAT CTT CTT G-3' (配列番号2)を含む前記8に記載の迅速判定キット。
[10] 制限酵素Bst4CIを含む前記9に記載の迅速判定キット。
[11]ヒトC反応性タンパク質遺伝子のSNP識別番号rs1205の塩基を解析するための核酸であって、ヒトC反応性タンパク質遺伝子のSNP識別番号rs1205の塩基を含み、かつ配列番号1及び配列番号2のプライマーを用いたPCR法によって増幅され得る領域に由来するDNA断片に対して特異的にハイブリダイズする核酸。
[12]ヒトC反応性タンパク質遺伝子の遺伝子多型を同定する際に使用する試料が、全血、白血球、ガン原発巣、リンパ管、リンパ節組織からなる群より選ばれる、前記1~7に記載の方法。
本発明で用いるCRP遺伝子はCRP(C-reactive protein:C反応性タンパク質)に対応する遺伝子である。CRPは、炎症に反応して主として肝細胞によって産生される急性期タンパク質の一種であり、従来、その血清中濃度は、様々な急性または慢性の炎症性疾患のマーカーとして用いられている。その名称は肺炎双球菌のC多糖体と沈降反応を示す血清タンパク質(βグロブリン分画に存在する)であることに由来し、感染、炎症、組織損傷などによって血中濃度が0.2μg/mLから数百倍から千倍に激増する。CRPは、分子量が約13万で、同一サブユニット5個からなる5量体であり、そのアミノ酸配列は血清アミロイドのPタンパク質、補体C1の一部と相同性がある。
[表A]
1 taaggcaaga gatctaggac ttctagcccc tgaactttca gccgaataca tcttttccaa
61 aggagtgaat tcaggccctt gtatcactgg cagcaggacg tgaccatgga gaagctgttg
121 tgtttcttgg tcttgaccag cctctctcat gcttttggcc agacaggtaa gggccacccc
181 aggctatggg agagatttga tctgaggtat gggggtgggg tctaagactg catgaacagt
241 ctcaaaaaaa aaaaaaaaag actgtatgaa cagaacagtg gagcatcctt catggtgtgt
301 gtgtgtgtgt gtgtgtgtgt gtgtgtgtgt gtggtgtgta actggagaag gggtcagtct
361 gtttctcaat cttaaattct atacgtaagt gaggggatag atctgtgtga tctgagaaac
421 ctctcacatt tgcttgtttt tctggctcac agacatgtcg aggaaggctt ttgtgtttcc
481 caaagagtcg gatacttcct atgtatccct caaagcaccg ttaacgaagc ctctcaaagc
541 cttcactgtg tgcctccact tctacacgga actgtcctcg acccgtgggt acagtatttt
601 ctcgtatgcc accaagagac aagacaatga gattctcata ttttggtcta aggatatagg
661 atacagtttt acagtgggtg ggtctgaaat attattcgag gttcctgaag tcacagtagc
721 tccagtacac atttgtacaa gctgggagtc cgcctcaggg atcgtggagt tctgggtaga
781 tgggaagccc agggtgagga agagtctgaa gaagggatac actgtggggg cagaagcaag
841 catcatcttg gggcaggagc aggattcctt cggtgggaac tttgaaggaa gccagtccct
901 ggtgggagac attggaaatg tgaacatgtg ggactttgtg ctgtcaccag atgagattaa
961 caccatctat cttggcgggc ccttcagtcc taatgtcctg aactggcggg cactgaagta
1021 tgaagtgcaa ggcgaagtgt tcaccaaacc ccagctgtgg ccctgaggcc cagctgtggg
1081 tcctgaaggt acctcccggt tttttacacc gcatgggccc cacgtctctg tctctggtac
1141 ctcccgcttt tttacactgc atggttccca cgtctctgtc tctgggcctt tgttccccta
1201 tatgcattgc aggcctgctc caccctcctc agcgcctgag aatggaggta aagtgtctgg
1261 tctgggagct cgttaactat gctgggaaac ggtccaaaag aatcagaatt tgaggtgttt
1321 tgttttcatt tttatttcaa gttggacaga tcttggagat aatttcttac ctcacataga
1381 tgagaaaact aacacccaga aaggagaaat gatgttataa aaaactcata aggcaagagc
1441 tgagaaggaa gcgctgatct tctatttaat tccccaccca tgacccccag aaagcaggag
1501 ggcattgccc acattcacag ggctcttcag tctcagaatc aggacactgg ccaggtgtct
1561 ggtttgggtc cagagtgctc atcatcatgt catagaactg ctgggcccag gtctcctgaa
1621 atgggaagcc cagcaatacc acgcagtccc tccactttct caaagcacac tggaaaggcc
1681 attagaattg ccccagcaga gcagatctgc tttttttcca gagcaaaatg aagcactagg
1741 tataaatatg ttgttactgc caagaactta aatgactggt ttttgtttgc ttgcagtgct
1801 ttcttaattt tatggctctt ctgggaaact cctccccttt tccacacgaa ccttgtgggg
1861 ctgtgaattc tttcttcatc cccgcattcc caatataccc aggccacaag agtggacgtg
1921 aaccacaggg tgtcctgtca gaggagccca tctcccatct ccccagctcc ctatctggag
1981 gatagttgga tagttacgtg ttcctagcag gaccaactac agtcttccca aggattgagt
2041 tatggacttt gggagtgaga catcttcttg ctgctggatt tccaagctga gaggacgtga
2101 acctgggacc accagtagcc atcttgtttg ccacatggag agagactrtg aggacagaag
2161 ccaaactgga agtggaggag ccaagggatt gacaaacaac agagccttga ccacgtggag
2221 tctctgaatc agccttgtct ggaaccagat ctacacctgg actgcccagg tctataagcc
2281 aataaagccc ctgtttactt g
[表B]
1 CTTTAGTTTT TGCTCCTCAA ATTGGAATAA TGATAGAATG AGAGTACTAA AACCCCCACA
61 ACTGGCCCTA CATGAATGGC CAGCTATCTC AAAAGAGGGA CTGTGCTTGT CAGAGGGAAT
121 CCCTTCAGGG GACTCTTGGA CAGGTTAAAG TGCCATGGAT ATGTTGTGTA ATGGGAAGTG
181 TAAACTTACA GGGACTTGAT TTCAAAGGTC ATTAGAGAAG TTAGCCACAA CTTCTAAAGC
241 AACTATCAGA AAACAGCTTG GACTCACTCA AGTAAACAGG GGCTTTATTG GCTTATAGAC
301 CTGGGCAGTC CAGGTGTAGA TCTGGTTCCA GACAAGGCTG ATTCAGAGAC TCCACGTGGT
361 CAAGGCTCTG TTGTTTGTCA ATCCCTTGGC TCCTCCACTT CCAGTTTGGC TTCTGTCCTC
421 AYAGTCTCTC TCCATGTGGC AAACAAGATG GCTACTGGTG GTCCCAGGTT CACGTCCTCT
481 CAGCTTGGAA ATCCAGCAGC AAGAAGATGT CTCACTCCCA AAGTCCATAA CTCAATCCTT
541 GGGAAGACTG TAGTTGGTCC TGCTAGGAAC ACGTAACTAT CCAACTATCC TCCAGATAGG
601 GAGCTGGGGA GATGGGAGAT GGGCTCCTCT GACAGGACAC CCTGTGGTTC ACGTCCACTC
661 TTGTGGCCTG GGTATATTGG GAATGCGGGG ATGAAGAAAG AATTCACAGC CCCACAAGGT
721 TCGTGTGGAA AAGGGGAGGA GTTTCCCAGA AGAGCCATAA AATTAAGAAA GCACTGCAAG
781 CAAACAAAAA CCAGTCATTT AAGTTCTTGG CAGTAACAAC ATATTTATAC CTAGTGCTTC
841 ATTTTGCTCT GGAAAAAAAG CAGATCTGCT CTGCTGGGGC AATTCTAATG GCCTTTCCAG
901 TGTGCTTTGA GAAAGTGGAG G
本発明はこの塩基の型が、C/C(ワイルド)、C/T(ヘテロ)、T/T(ホモ)のいずれのタイプであるかを同定することにより、ガンのリンパ節転移又はそのリスクを判定するものである。
SNP識別番号rs1205は、米国のNCBI(National Center for Biotechnology Information)のSNPデータベース(dbSNP)に登録されているSNPの識別番号であり、rs1205で登録されているSNPの情報はNCBIのサイト(http://www.ncbi.nlm.nih.gov/projects/SNP/)から入手することができる。
上述した同定方法のうち、被験DNAが少量の場合に好適な方法、および多数の被験DNAについて同定する場合に好適な方法についてそれぞれ代表的な方法を例に説明する。
TaqMan PCR法とは、蛍光標識したアレル特異的オリゴとTaq DNAポリメラーゼによるPCR反応とを利用した方法である。
当業者に周知の方法によって本発明のリンパ節転移又はそのリスクの迅速判定キットを調製することができる。キットの形態として、例えば、本発明のプライマーを用いてCRP遺伝子多型の検出を行う際に必要な各種の試薬類は、予めパッケージングしてキット化することができる。具体的には、本発明のプライマーあるいはループプライマーとして必要な各種のオリゴヌクレオチド、核酸合成の基質となる4種類のdNTP(dATP、dCTP、dGTP及びdTTP)、鎖置換活性を有する上記の鋳型依存性核酸合成酵素、酵素反応に好適な条件を与える緩衝液、補助因子としての塩類(マグネシウム塩又はマンガン塩等)、酵素や鋳型を安定化する保護剤、さらに制限酵素、及び必要に応じて反応生成物の検出に必要な試薬類がキットとして提供される。また、標的核酸とハイブリダイズし得るプローブDNAを構成試薬としてキット化してもよい。
この例は、胸部食道扁平上皮(細胞)癌ガン患者113名(すべて日本人)について行なった。うち38名は、病理学的診断等により食道ガンを確認した上で2007年4月から1年間の間に食道切除を行なった。残る75名は2000年~2007年の間に食道切除術を施術し、その後の経過の観察を行なった者から無作為に抽出した。疾患の分類は国際対ガン協会による腫瘍リンパ節転移(TNM)分類第6版により行なった。
対照として秋田大学病院内においてガン以外の疾病で処置を受けている139名(すべて日本人)のCRP多型(CRP1846C>T(rs1205)遺伝子多型及び上述の3種類の遺伝子多型の計4種類)も調べた。
多型の出現頻度は、ハーディ・ワインベルグ平衡にあると考えた場合と矛盾しない。また、国立がんセンターにおけるSNP500データベースとも同様の結果であった。
同様に152例の肺癌手術患者(すべて日本人)に関してCRP1846C>T(rs1205)遺伝子多型と病理学的リンパ節転移の関連を検討した結果、食道癌同様に有意な関連を認めた(Fisherの正確検定, P=0.0312)。
実施例1の患者113名中、壁深達度がpT1-2である64名についてCRP1846C>T(rs1205)遺伝子多型とリンパ節転移との関係を解析した。CRP1846C>T(rs1205)遺伝子多型においてC/CおよびC/T遺伝子タイプ患者でリンパ節転移を認めたのは6例、転移を認めなかったのは35例であるのに対し、T/T遺伝子タイプ患者ではリンパ節転移を認めたのは18例、転移を認めなかったのは5例であり、CRP1846C>T(rs1205) 遺伝子多型とリンパ節転移とは有意に関連した(Fisherの正確検定, P=0.0001)。このように本発明方法では、従来の画像診断方法では見落とされる可能性が考えられる、より早期の癌においても確実にリンパ節転移を判定することができるため、臨床の要求に応え得ることが確認された。
実施例2の患者152名中、リンパ管侵襲に関する所見記載のあった144名についてCRP1846C>T(rs1205)遺伝子多型とリンパ管侵襲との関係を解析した。CRP1846(rs1205)遺伝子多型においてC/CおよびC/T遺伝子タイプ患者でリンパ管侵襲を認めたのは36例、リンパ管侵襲を認めなかったのは42例であるのに対し、T/T遺伝子タイプ患者ではリンパ管侵襲を認めたのは45例、リンパ管侵襲を認めなかったのは21例であり、CRP1846C>T(rs1205)遺伝子多型とリンパ管侵襲とは有意に関連した(Fisherの正確検定, P=0.008)。「リンパ管侵襲」は原発部位のリンパ管にガン細胞の存在が観察された状態を指し、現に「リンパ節転移」がない場合でも、将来の「リンパ節転移」の可能性を示唆している。従って、「リンパ節転移」のリスク判定(より早期の予知)にCRP1846C>T(rs1205)遺伝子多型の同定は有用である。
Claims (12)
- ヒトC反応性タンパク質遺伝子の遺伝子多型を同定することにより、ガンのリンパ節転移またはそのリスクを判定する方法。
- SNP識別番号rs1205の遺伝子多型を同定することにより、ガンのリンパ節転移またはそのリスクを判定する請求項1に記載の方法。
- SNP識別番号rs1205の遺伝子多型がT/Tタイプであるときに高リスクと判定する請求項2に記載の方法。
- 遺伝子多型の同定をRFLPまたは対応する相補鎖配列との結合により行なう請求項1~3のいずれかに記載の方法。
- 遺伝子多型の同定をPCR-RFLPにより行なう請求項4に記載の方法。
- PCRにおけるプライマーとして Forwardプライマー:5'-CTT ATA GAC CTG GGC AGT-3'(配列番号1)、Reverseプライマー:5'-GGA GTG AGA CAT CTT CTT G-3' (配列番号2)を用い、制限酵素としてBst4CIを用いる請求項5に記載の方法。
- ガンが固形ガンである請求項1~6のいずれかに記載の方法。
- ヒトC反応性タンパク質遺伝子の塩基配列のSNP識別番号rs1205を含む領域を増幅するためのプライマーとRFLPによりSNP識別番号rs1205の遺伝子多型を判定するための制限酵素を含むガンのリンパ節転移またはそのリスクを判定するためのPCR-RFLP用迅速判定キット。
- プライマー対としてForwardプライマー:5'-CTT ATA GAC CTG GGC AGT-3' (配列番号1)、Reverseプライマー:5'-GGA GTG AGA CAT CTT CTT G-3' (配列番号2)を含む請求項8に記載の迅速判定キット。
- 制限酵素Bst4CIを含む請求項9に記載の迅速判定キット。
- ヒトC反応性タンパク質遺伝子のSNP識別番号rs1205の塩基を解析するための核酸であって、ヒトC反応性タンパク質遺伝子のSNP識別番号rs1205の塩基を含み、かつ配列番号1及び配列番号2のプライマーを用いたPCR法によって増幅され得る領域に由来するDNA断片に対して特異的にハイブリダイズする核酸。
- ヒトC反応性タンパク質遺伝子の遺伝子多型を同定する際に使用する試料が、全血、白血球、ガン原発巣、リンパ管、リンパ節組織からなる群より選ばれる、請求項1~7に記載の方法。
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WO2014119556A1 (ja) * | 2013-01-30 | 2014-08-07 | 国立大学法人秋田大学 | ガンのリンパ節転移抑制剤 |
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US20150292037A1 (en) | 2015-10-15 |
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