# Patent

## Title
Method for determining lymph node metastasis in cancer or risk thereof and rapid determination kit for the same

## Abstract

The objective of the present invention is to provide a method and a means of rapidly and reliably detecting lymph node metastasis in cancer or the risk of lymph node metastasis. Specifically, the present invention provides a method and a rapid determination kit for detecting lymph node metastasis in cancer or its risk by identifying a certain genetic polymorphism of the human CRP gene, and it is clinically significant in determining the treatment strategy, because effective prediction/determination can be made regarding lymph node metastasis, which is an important phenomenon in cancer progression.

## Background
TECHNICAL FIELD
The present invention relates to a method for determining lymph node metastasis in cancer and a rapid determination kit for the same.
BACKGROUND ART
Cancer cells from a primary focus metastasize all over the body through blood vessels and lymphatic vessels. Because the primary focus is removed as much as possible in cancer surgery, the accurate detection of metastasis and the appropriate treatment depending on the degree of metastasis are necessary. Therefore, diagnosing lymph node metastasis by cancer cells is extremely important for selecting the appropriate treatment for cancer.
The diagnosis of lymph node metastasis by cancer cells is broadly divided into pretreatment diagnostic imaging and posttreatment (postoperative) pathological diagnosis. The diagnostic imaging methods that are used for detecting lymph node metastasis in cancer (inspection for the presence of lymph node metastasis) include computed tomography (CT); positron emission tomography (PET); PET-CT, which uses an apparatus integrating PET and CT; and endoscopic ultrasoundscopy (EUS); however, the diagnostic imaging methods have difficulty or only limited availability in detecting microscopic lymph node metastasis. On the other hand, pathological diagnosis method utilizes specimens prepared from a number of excised lymph node tissues under a microscope and is a highly accurate and reliable diagnostic method; however, the diagnosis can only be made using excised lymph nodes as posttreatment (postoperative) diagnosis and therefore cannot be used for selecting the optimum treatment in advance. The diagnosis of lymph node metastasis by cancer cells is problematic in that the pretreatment diagnosis depends on the diagnostic imaging, which is currently less accurate, whereas reliable diagnosis is made in the posttreatment (postoperative) pathological diagnosis.
Therefore, molecular diagnostic techniques using molecular markers are important in the diagnosis of lymph node metastasis by cancer cells, and several techniques have been developed. Many conventionally known molecular diagnostic techniques utilize a protein (target protein) that is not expressed or expressed at a lower level in normal cells and is highly expressed in cancer cells or a nucleic acid (target nucleic acid, as a general term for DNA, mRNA, cDNA, etc.) included in a gene encoding the target protein. Specifically, a target protein included in lymph node tissues resected/excised from a living body is detected using an immunoassay, or conversely, a target nucleic acid is amplified using loop-mediated isothermal amplification (LAMP) or polymerase chain reaction (PCR) to detect the amplification product using a known method to determine the presence of metastatic cancer cells.
Regarding molecular diagnostic techniques, for example, Patent Literature 1 (Japanese Laid-Open Patent Publication No. 2007-175021) proposed a method for determining the presence of lymph node metastasis by colon cancer cells using the mRNA or a fragment of a gene encoding at least one protein selected from the group consisting of PIGR, CLDN3, LGALS4, AGR2, TACSTD1, GPX2, RAI3, TSPAN1, CKB, ELF3, FXYD3, CDH1, REG4, GDF 15, CLDN4, OLFM 4, CD9, CDH17, SELENBP, LCN2, TMPRSS4, CFTR, TM4SF3, ID1, CYP2S1, TFF3, EHF, FAT, KLF5, SLC9A3R2, HOXB9, ATP1B1, PCK1, and FCGBP. Patent Literature 2 (Japanese Laid-Open Patent Publication No. 2007-037421) described the determination of lymph node metastasis in colon cancer by entering the value of expression of a gene set represented by the database access numbers (serial numbers) NM—003404 (G1592), NM—002128 (G2645), NM—052868 (G3031), NM—005034 (G3177), NM—001540 (G3753), NM—005722 (G3826), and NM—015315(G4370) into a mathematical function. Patent Literature 3 (Japanese Laid-Open Patent Publication No. 2008-020438) describes that lymph node metastasis, e.g., from breast cancer, can be determined with higher reliability by determining the expression of a polypeptide related to cytokeratin in a sample prepared from lymph node tissue.
On the other hand, it was recently determined that inflammatory responses promote carcinogenesis by damaging DNA, stimulating angiogenesis and cell proliferation, and inhibiting apoptosis. In this regard, serum C-reactive protein (CRP) has been investigated as a risk factor and a prognostic factor in colon (Non Patent Literature 1: Erlinger T. P. et al., JAMA 2004; 291; 585-590), esophageal (Non Patent Literature 2: Shimada H. et al., J. Surg. Oncol. 2003; 83; 248-252), hepatocellular (Non Patent Literature 3: Hashimoto K. et al., Cancer 2005; 103; 1856-1864), renal (Non Patent Literature 4: Miyata Y. et al., Urology 2001; 58; 161-164), and ovarian (Non Patent Literature 5: Hefler L. A. et al., Clin. Cancer Res. 2008; 14; 710-714) cancers.
A higher serum CRP level is considered to be associated with a higher risk of developing cancer. For example, Non Patent Literature 6 (Nozoe T. et al., Am. J. Surg. 1998; 176(4):335-8) describes that liver metastasis and lymph node metastasis in colon cancer patients are associated with preoperative increases in serum CRP levels, Non Patent Literature 7 (Nozoe T. et al., Am. J. Surg. 2001; 182(2), 197-201) describes that lymph node metastasis in esophageal cancer patients is associated with preoperative increases in serum CRP levels, and Non Patent Literature 8 (Ines G. et al., World J. Gastroenterol. 2006; 12(23), 3746-3750) describes that a higher serum CRP level in esophageal cancer patients is associated with lymph node metastasis.
It has been reported that genetic polymorphisms are strongly related to serum CRP levels (Non Patent Literature 9: Carlson C. S. et al., Am. J. Hum. Gen. 2005; 77; 64-77 and Non Patent Literature 10: Szalai A. J. et al., J. Mol. Med. 2005; 83; 440-447).
Therefore, the present inventors examined whether CRP genetic polymorphisms act as cancer progression factors in esophageal cancer patients. Although we consequently revealed the potential association of CRP-717T>C genetic polymorphisms with lymph node metastasis (Non Patent Literature 11: Motoyama et al., The Japanese Journal of Gastroenterological Surgery, vol. 41, No. 7, pp. 1169, July 2008), a technique for detecting lymph node metastasis by cancer cells using the CRP-717T>C genetic polymorphism suffered from lower determination accuracy and was not put into practical use because metastasis was not statistically significant.
CITATION LIST
Patent Literature


    [Patent Literature 1] Japanese Laid-Open Patent Publication No. 2007-175021
    [Patent Literature 2] Japanese Laid-Open Patent Publication No. 2007-037421
    [Patent Literature 3] Japanese Laid-Open Patent Publication No. 2008-020438


Non Patent Literature


    [Non Patent Literature 1] Erlinger T. P. et al., JAMA 2004; 291; 585-590
    [Non Patent Literature 2] Shimada H. et al., J. Surg. Oncol. 2003; 83; 248-252
    [Non Patent Literature 3] Hashimoto K. et al., Cancer 2005; 103; 1856-1864
    [Non Patent Literature 4] Miyata Y. et al., Urology 2001; 58; 161-164
    [Non Patent Literature 5] Hefler L. A. et al., Clin. Cancer Res. 2008; 14; 710-714
    [Non Patent Literature 6] Nozoe T. et al., Am. J. Surg. 1998; 176(4):335-8
    [Non Patent Literature 7] Nozoe T. et al., Am. J. Surg. 2001; 182(2), 197-201
    [Non Patent Literature 8] Ines G. et al., World J. Gastroenterol. 2006; 12(23), 3746-3750
    [Non Patent Literature 9] Carlson C. S. et al., Am. J. Hum. Gen. 2005; 77; 64-77
    [Non Patent Literature 10] Szalai A. J. et al., J. Mol. Med. 2005; 83; 440-447
    [Non Patent Literature 11] Motoyama et al., The Japanese Journal of Gastroenterological Surgery vol. 41, No. 7, pp. 1169, July 2008


SUMMARY OF INVENTION
Technical Problem
Regarding methods to detect lymph node metastasis by cancer cells using a conventionally known molecular diagnostic technique, as described in the methods of Patent Literatures 1 to 3, an attempt to reliably detect lymph node metastasis requires a comprehensive investigation of multiple molecular markers as factors for determination and the necessity of using lymph nodes, which greatly torment patients and require considerable time and effort for sample preparation. Serum CRP levels often fluctuate due to the influences of age, smoking, and inflammation. Moreover, the association of the CRP-717T>C genetic polymorphism with lymph node metastasis by cancer cells, as described by Non Patent Literature 11, was later denied. Therefore, a new molecular marker for rapidly and reliably detecting lymph node metastasis by cancer cells and a molecular diagnostic technique using the molecular marker are desired.
Solution to Problem
As described above, a higher serum CRP level is associated with a certain CRP genetic polymorphism. However, the present inventors have found that the usage of the SNP rs1205 (also referred to as CRP1846C>T or rs1205 in this description) as a molecular marker dramatically improves the determination accuracy of lymph node metastasis in cancer and is extremely useful, which is not assumed from the conventional prediction that a CRP genetic polymorphism related to a high serum CRP level may act as a cancer progression factor, thereby completing the present invention. The SNP rs1205 is a one-base mutation in a nontranscribed region of the CRP gene that has been reported to be correlated with a decrease in serum CRP levels.
The present invention provides the following determination method and determination kit:
[1] A method of determining lymph node metastasis in cancer or the risk thereof by identifying a genetic polymorphism in the human C-reactive protein (CRP) gene
[2] The method according to item 1 above, wherein lymph node metastasis in cancer or the risk thereof is determined by identifying a genetic polymorphism SNP rs1205
[3] The method according to item 2 above, wherein the risk is considered to be high when the genotype of SNP rs1205 is T/T
[4] The method according to any one of items 1 to 3 above, wherein the genotype is identified by restriction fragment length polymorphism (RFLP) or by analyzing its binding to a corresponding complementary strand sequence
[5] The method according to item 4 above, wherein the genotype is identified by PCR-RFLP
[6] The method according to item 5 above, wherein a forward primer, 5′-CTT ATA GAC CTG GGC AGT-3′ (SEQ ID No. 1), and a reverse primer, 5′-GGA GTG AGA CAT CTT CTT G-3′ (SEQ ID No. 2), are used for PCR and Bst4CI is used as a restriction enzyme
[7] The method according to any one of items 1 to 6 above, wherein the cancer is a solid cancer
[8] A rapid determination kit for PCR-RFLP for detecting lymph node metastasis in cancer or the risk thereof, in which the kit comprises primers for amplifying a region containing SNP rs1205 of base sequence of human C-reactive protein gene and a restriction enzyme for detecting the genotype of SNP rs1205 by RFLP
[9] The rapid determination kit according to item 8 above, comprising a forward primer, 5′-CTT ATA GAC CTG GGC AGT-3′ (SEQ ID No. 1), and a reverse primer, 5′-GGA GTG AGA CAT CTT CTT G-3′ (SEQ ID No. 2), as a primer pair
[10] The rapid determination kit according to item 9 above, comprising the restriction enzyme Bst4CI
[11] A nucleic acid for analyzing bases of the SNP identification number rs1205 of human C-reactive protein gene, the nucleic acid specifically hybridizing to a DNA fragment derived from a region containing the bases of the SNP identification number rs1205 of the human C-reactive protein gene, the region being amplifiable by a PCR method using primers of SEQ ID Nos. 1 and 2.
[12] The method according to any one of items 1 to 7 above, wherein the sample used in identifying the genotype of the human CRP gene is selected from the group consisting of whole blood, leukocytes, the primary focus of cancer, lymphatic vessels, and lymph node tissue.
Advantageous Effects of Invention
Although it is known that the production of CRP is associated with various cytokines (interleukins, tumor necrosis factors, interferons, transforming growth factors), the method of the present invention is independent of the levels of various cytokines and can by itself effectively predict/detect lymph node metastasis by cancer cells. The method of detecting lymph node metastasis in cancer using SNP rs1205 is simple compared to conventional methods and yet remains extremely accurate. Therefore, the usage of SNP rs1205 enables the detection of lymph node metastasis with statistical significance. Because effective prediction/detection can be made on the basis of lymph node metastasis, which is an important phenomenon in cancer progression, the method enables the selection of the most reliable and least invasive therapy from options such as surgery involving lymph node dissection, endoscopic resection without lymph node dissection, chemoradiotherapy, chemotherapy, and radiation therapy, making the present invention clinically significant in determining the treatment strategy.
As the current diagnosis of lymph node metastasis of cancer cell has problems in that the pretreatment diagnosis depends on diagnostic imaging, which is currently less accurate, and that reliable diagnosis is as achieved only as a pathological diagnosis, which is a posttreatment (postoperative) diagnosis, the method of the present invention can solve both problems simultaneously.
Moreover, because this method does not require lymph tissue (lymph nodes or lymphatic vessel) to detect the presence of SNP rs1205 (peripheral blood and other substances/tissues can be used instead), patients suffer less, and work load of laboratory personnel is reduced because of the reduced requirements of sample preparation.


DESCRIPTION OF EMBODIMENTS
A. Method of Detecting Lymph Node Metastasis and the Risk Thereof
The CRP gene used in this invention is a gene corresponding to CRP (C-reactive protein). CRP is a type of acute-phase protein produced predominantly by hepatocytes in response to inflammation, and its serum levels are conventionally used as a marker for various acute and chronic inflammatory diseases. The name is derived from a serum protein (present in the β-globulin fraction) causing a precipitation reaction with the C-polysaccharide of Diplococcus pneumoniae, and its expression drastically increases in blood from 0.2 μg/mL by a factor of several hundred to one thousand due to infection, inflammation, and tissue damage. CRP is a homopentamer having a molecular weight of one hundred thirty thousand daltons, and its amino acid sequence is homologous to a portion of serum amyloid P-protein, complement C1.
The entire sequence of the CRP gene has been identified (Woo P, Korenberg J R, Whitehead A S, J. Biol. Chem., 260:13384-13388, 1985) and can be retrieved, for example, as Accession No. NG—013007 in the NCBI web site (http://www.ncbi.nlm.nih.gov/nuccore/NG—013007.1?report=gbwithparts&log$=seqview&from=5 000&to=7300) (see Table A for the entire base sequence)(SEQ ID NO: 3).








TABLE 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









In genetics, a genetic polymorphism is normally defined as an alteration (mutation) of certain bases present in one gene at a frequency of 1% or more in the population. A number of genetic polymorphisms have been identified in the CRP gene, and the genetic polymorphism SNP rs1205 can preferably be utilized in the present invention. SNP rs1205 is a polymorphism in the 2148th nucleotide (base denoted by r) of the CRP gene sequence described in Table A. In this sequence, r indicates that the base is G (guanine) or A (adenine).
Table B (SEQ ID NO: 4) describes the sequence around SNP rs1205 in a sequence complementary to the CRP gene sequence described in Table A.








TABLE 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









SNP rs1205 denotes a polymorphism in the 422nd base (base denoted by Y) of the base sequence described in Table B. In this sequence, Y indicates that the base is C (cytosine) or T (thymine).
In the present invention, lymph node metastasis in cancer or the risk thereof is determined by identifying the genotype of this base (C/C (wild-type), C/T (heterozygous), and T/T (homozygous)).
rs1205 is the identification number of the SNP registered in the SNP database of the National Center for Biotechnology Information (dbSNP, NCBI) in the US, and information about rs1205 is available from the NCBI website (http://www.ncbi.nlm.nih.gov/projects/SNP/).
SNP rs1205 can be identified with various known methods capable of detecting genetic polymorphisms, such as polymerase chain reaction (PCR), PCR-restriction fragment length polymorphism (PCR-RFLP), PCR-single strand conformation polymorphism (PCR-SSCP) (e.g., Orita, M. et al., Proc. Natl. Acad. Sci., U.S.A., 86, 2766-2770 (1989)), PCR-specific sequence oligonucleotide (PCR-SSO), an allele-specific oligonucleotide (ASO) hybridization method (e.g., Saiki, Nature, 324, 163-166 (1986)) combining the PCR-SSO method and a dot hybridization method, Taq-Man-PCR (Livak, K J, Genet Anal., 14, 143 (1999), Morris, T. et al., J. Clin. Microbiol., 34, 2933 (1996)), an Invader method (Lyamichev et al., Nat. Biotechnol., 17, 292 (1999)), a MALDI-TOF/MS (matrix) method (Haff L A, Smirnov I P, Genome Res., 7, 378 (1997)) using primer extension, a rolling circle amplification (RCA) method (Lizardi P M et al., Nat Genet 19, 225 (1998)), a method using DNA chip or microarray (e.g., Wang D G et al., Science 280,1077 (1998)), primer extension, a Southern blot hybridization method, and a dot hybridization method (e.g., Southern, E., J. Mol. Biol. 98, 503-517 (1975)), and this identification is not particularly limited. The corresponding sequence portion may be analyzed by direct sequencing. These methods can be used in arbitrary combinations. When the method is implemented, primers and probes can be designed from the base sequences described in Tables A and B as needed.
When the identification method is implemented, if the number of target DNA is small, it is preferable to use PCR-based method, e.g., PCR-RFLP, for the identification in terms of detection sensitivity and accuracy. Any of the identification methods may be applied after the test DNA is amplified in advance by PCR or a PCR-based gene amplification method. Conversely, if the identification is performed for a number of target DNA, it is particularly preferable to use methods incorporating DNA chips or microarrays, the Invader method, TaqMan-PCR, and the MALDI-TOF/MS (matrix) method using primer extension or RCA method.
Among the identification methods described above, the preferred method in the case of a small number of target DNA and the preferred method in the case of identification for a large number of target DNA will be described by using representative methods as examples.
The preferred method in the case of a small number of target DNA is preparing a DNA sample from a patient with a method well known to those skilled in the art, followed by cleaving the prepared DNA sample with a restriction enzyme, separating DNA fragments depending on their size, and then comparing the sizes of the detected DNA fragments with that of a control. Typically, a DNA sample is first prepared from a patient and DNA containing the CRP gene is then amplified. The amplified DNA is cleaved by restriction enzyme. DNA fragments are then separated depending on its size and the size of detected DNA fragment is compared with a control.
Such methods include RFLP and PCR-RFLP. In other words, if mutation exists in the recognition site of a restriction enzyme or if a base insertion or deletion exists in a DNA fragment generated by a restriction enzyme, the size of the fragment generated after restriction enzyme treatment is changed compared to that of the control. The portions including this mutation can be amplified by PCR and treated with respective restriction enzymes to detect the mutation as a difference in the mobility of the bands after electrophoresis. Alternatively, after a DNA sample (genomic (chromosomal) DNA is usable) is prepared from a patient, treated with restriction enzymes, and electrophoresed, the presence of a polymorphism (mutation) can be detected by Southern blotting using a DNA probe that may hybridize with a target nucleic acid. The restriction enzymes used can be selected depending on the respective mutations as needed. In this method, in addition to genomic DNA, RNA prepared from patients can be converted into cDNA using reverse transcriptase and directly cleaved with a restriction enzyme to perform Southern blotting. DNA containing the CRP gene can be amplified by PCR using this cDNA as a template and cleaved with a restriction enzyme to examine differences in mobility.
Primers used in the present invention include all of the primers that can amplify DNA containing the CRP gene. The preferred base length of the primers is 10 or more bases, most preferably 15 or more bases. Each of the primers may be a single oligonucleotide or a mixture of a number of oligonucleotides. Examples of the primers used in PCR are a forward primer, 5′-CTT ATA GAC CTG GGC AGT-3′ (SEQ ID No. 1), and a reverse primer, 5′-GGA GTG AGA CAT CTT CTT G-3′ (SEQ ID No. 2). The restriction enzyme may be Bst4CI. Materials other than the primers and conditions in PCR, application of the restriction enzyme, electrophoresis, detection, and other conditions may be the same as those of commonly used methods.
The DNA probes that can be used in Southern blotting are not particularly limited, provided that the DNA probe can hybridize with the target nucleic acid. An example of a DNA probe hybridizable to the target nucleic acid is that for SNP rs1205 of the human CRP gene, which specifically hybridizes with a DNA fragment containing rs1205 and which is derived from a region amplifiable by PCR using SEQ ID Nos. 1 and 2.
The CRP gene can be acquired from blood cells, peripheral blood leukocytes, skin cells, mucosal cells, liver, kidney, adrenal gland, brain, and uterine tissues, hair, and other tissues of patients by using known extraction and purification methods. A partial- or full-length sequence of the CRP gene may be utilized in the present invention as long as it contains the target base. In other words, a DNA fragment of any length is usable as long as it contains SNP rs1205.
The preferred method in the case of identification for a large number of target DNA is preparing DNA containing the CRP gene derived from a patient and a substrate fixed to nucleotide probes (synonymous with the DNA probes) hybridizing to the DNA, followed by bringing the DNA into contact with the substrate and subsequently detecting DNA (target nucleic acid) hybridizing to the nucleotide probes fixed to the substrate to detect PCR genetic polymorphisms.
Such a method can be well illustrated by a DNA chip method (microarray method). A DNA sample from a patient containing the CRP gene can be prepared with a method well known to those skilled in the art as described above. In the preferred method of DNA sample preparation, DNA is prepared from genomic (chromosomal) DNA extracted from blood, peripheral blood leukocyte, cells such as skin cell, and mucosal cell, tissues such as liver, kidney, adrenal gland, brain, and uterus, hair of patients as described above. To prepare a DNA sample of this method from genomic (chromosomal) DNA, for example, a primer hybridizing to DNA containing the CRP gene can be used for preparing DNA containing the CRP gene with PCR using the genomic (chromosomal) DNA as a template. The prepared DNA sample can be labeled as needed for detection with a method well known to those skilled in the art.
In the DNA chip method, a plurality of DNA probes is aligned and fixed on a substrate of glass or another material to perform hybridization of a labeled DNA sample, and a method of detecting a label (such as fluorescence) signal on the probe is utilized to distinguish and detect complete matches and one-base mismatches through hybridization to detect genetic polymorphisms such as SNPs.
The preferred methods in the case of identification for a large number of target DNA will hereinafter be summarized.
The TaqMan PCR method utilizes PCR of fluorescently labeled allele-specific oligos and Taq DNA polymerase.
The Invader method is the combination of i) the hybridization to template DNAs of two reporter probes specific to respective alleles of genetic polymorphisms such as SNPs and one invader probe, and ii) the cleavage of DNA by an enzyme exhibiting special endonuclease activity that recognizes and cleaves a specific structure of DNA.
The SniPer method can be employed as a method utilizing primer extension. A basic principle of the SniPer method is a technique called rolling circle amplification (RCA), and circular single-stranded DNA is used as a template for DNA polymerase to continuously synthesize complementary-strand DNA. With this method, genetic polymorphisms such as SNPs can be determined by measuring the presence of a color reaction generated when DNA amplification occurs.
The MALDI-TOF/MS method utilizes a mass spectrometer to detect changes in mass due to one-base substitution for genotyping SNPs. Methods utilizing PCR amplification and multiplex PCR are available.
The sequencing method can be used to amplify a region containing a genetic polymorphism using PCR and a dye terminator to sequence DNA to analyze the frequency of genetic polymorphisms such as SNPs.
The determination method of the present invention is applicable to various stages and is particularly useful in the decision of the treatment strategy. For example, for patients with esophageal cancer with submucosal invasion, detection of lymph node metastasis using conventional methods is difficult. On the other hand, the present invention can detect lymph node metastasis or the risk thereof with high accuracy and can therefore avoid the deterioration of QOL due to unnecessary lymph node dissection and prevent cancer from progressing without necessary lymph node dissection.
No particular limitation exists on the types of cancer for which this method is applicable, and the method is applicable to all the solid cancers. Specifically, the method is applicable to cancers with primary focus in the esophagus, lung, breast, head and neck, stomach, colon, biliary tract, pancreas, uterus, ovary, bladder, kidney, urothelium, and prostate gland.
B. Rapid Determination Kit for Lymph Node Metastasis or the Risk Thereof
The present rapid determination kit for lymph node metastasis and the risk thereof can be prepared with a method well known to those skilled in the art. Various reagents necessary for detecting the CRP genetic polymorphism by using the primers of the present invention can be packaged in advance into the kit. Specifically, the reagents provided as a kit include various oligonucleotides used as the primers or loop primers designed for the present invention, four types of dNTP (dATP, dCTP, dGTP, and dTTP) acting as substrates for nucleic acid synthesis, the template-dependent nucleic acid synthesis enzyme exhibiting strand displacement activity, buffer solution providing preferred conditions for the enzyme reaction, salts (e.g., magnesium salt or manganese salt) as cofactors, protectants stabilizing enzymes and templates, and restriction enzyme as well as reagents necessary for detecting reaction products as needed. DNA probes hybridizable to the target nucleic acid may be included in a kit as a component reagent.
Although terms used in this description have normally used meanings, “lymph node” in the “method of detecting lymph node metastasis in cancer or the risk thereof” may be used as a general term for both “lymph node” and “lymphatic vessel” or a term for “lymphoid tissue”; “detection of lymph node metastasis” includes detecting the presence/possibility of cancer cells existing in a lymph node; and “determination of the risk of lymph node metastasis” includes determining the presence/possibility of metastasis of cancer cells from a primary focus to a lymph node if a certain individual has a cancer.
EXAMPLES
Although the present invention will hereinafter be described in more detail with examples, it is not limited to these examples. The following study including experiments/examinations was performed with the approval from the Ethics Committee of Akita University School of Medicine. All the subjects provided informed consent.
Example 1
This example was performed using 113 patients (all Japanese) with thoracic esophageal squamous cell cancer. Thirty-eight of the patients underwent esophagectomy over 1 year beginning in April 2007 after confirmation (e.g., by pathological diagnosis) of esophageal cancer. The remaining 75 patients were selected at random from among those undergoing esophagectomy between 2000 and 2007 and were observed for subsequent cancer progression. The disease was classified in accordance with the International Union against Cancer Tumor-Node-Metastasis (TNM) classification of malignant tumors, 6th edition.
After collecting peripheral blood from the patients, DNA was extracted using a QIAamp Blood Kit (Qiagen) and stored at −80° C. until analysis. The investigation of the association with lymph node metastasis in cancer was performed for CRP1846C>T(rs1205), the example of the present invention, and for 18 other genetic polymorphisms, i.e., CRP polymorphisms CRP-717C>T(rs2794521), CRP1059G>C(rs1800947), and CRP1444C>T(rs1130864); tumor necrosis factor polymorphisms TNF-α-238G>A, TNF-α-308G>A, TNF-α-1031T>C; and TNF-β250G>A, INF-γ874A>T, TGF-β1 29T>C, IL-1β-31C>T, IL-1β-511C>T, IL-1 receptor antagonist, IL-2-330T>G, IL-4-590C>T, IL-6-634G>C, IL-6 receptor 48892A>C, IL-10-592A>C, and IL-12β-1188A>C.
PCR for amplifying target nucleic acids was performed by thermal denaturation of extracted DNA at 95° C. for 15 min, 35 cycles of reaction at 95° C. for 30 s, 56° C. for 30 s, and 72° C. for 30 s, and heating at 72° C. for 5 min. A forward primer, 5′-CTT ATA GAC CTG GGC AGT-3′ (SEQ ID No. 1), and a reverse primer, 5′-GGA GTG AGA CAT CTT CTT G-3′ (SEQ ID No. 2), were used as primers for amplifying the CRP1846C>T (rs1205) genetic polymorphism. Bst4CI was added to the PCR amplification product acquired from the operation, and after incubation at 65° C. for 8 h, RFLP was performed by electrophoresis.
CRP polymorphisms (a total of four genetic polymorphisms, i.e., the CRP1846C>T (rs1205) genetic polymorphism and three genetic polymorphisms mentioned above) were also investigated in 139 patients (all Japanese) treated for ailments other than cancer in Akita University Hospital as controls.
The frequencies of the appearance of the polymorphisms are consistent with those expected for Hardy-Weinberg equilibrium. The results were similar to those of the SNP500 database of the National Cancer Institute.
Of the 113 esophageal cancer patients studied, 62 patients (55%) had lymph node metastasis, whereas 51 patients (45%) did not have lymph node metastasis. Although patients with pathologically identified lymph node metastasis had significantly (P<0.05) deeper invasion by the cancer than the patients without lymph node metastasis, no significant correlation existed between the presence of lymph node metastasis and age, sex, preoperative nutritional state, tumor markers, tumor site and size, squamous cell and intramural metastasis (see Table 1 “Clinical characteristics of patients with or without lymph node metastasis”).







TABLE 1







Clinical characteristics of patients


with or without lymph node metastasis










Lymph node metastasis












Negative
Positive




(N = 51)
(N = 62)
P














Age (years)
65 ± 8 
63 ± 8 
0.106


Gender


Male
47
52


Female
 4
10
0.2538


Hemoglobin (g/dL)
13.6 ± 1.5 
13.7 ± 1.5 
0.7718


Albumin (g/L)
43 ± 3 
43 ± 3 
0.4647


SCC (ng/mL)
1.1 ± 2.9
1.1 ± 1.5
0.8970


CEA (ng/mL)
3.5 ± 2.1
4.3 ± 2.8
0.1022


Preoperative serum
4.0 ± 8.3
4.7 ± 8.1
0.6514


CRP (mg/L)


Tumor location


Upper third
 2
 5


Middle third
31
31


Lower third
28
26
0.4305


Tumor size (mm)
49 ± 29
54 ± 25
0.3725


Depth of tumor


invasion (pT)


T1
27
14


T2
 4
11


T3
18
31


T4
 2
 6
0.0078*


Tumor differentiation


Well-moderately
41
48


Poorly
10
14
0.8184


Lymphatic invasion


Positive
45
62


Negative
 6
 0
0.0071*


Venous invasion


Positive
34
55


Negative
17
 7


Intramural metastasis


Positive
 4
 8


Negative
47
54
0.5422


Number of involved
 0
3.9 ± 4.9
<0.001*


lymph nodes





SCC squamous cell carcinoma antigen, CEA carcinoembryonic antigen, CRP C-reactive protein






As a result of the analysis of the association between various genetic polymorphisms and pathologically identified lymph node metastasis, only the CRP1846C>T (rs1205) genetic polymorphism which is utilized in the present invention was significantly associated with lymph node metastasis (Fisher's exact test, P=0.0043). Regarding the CRP1846C>T (rs1205) genetic polymorphism, lymph node metastasis was found in 25 patients and not found in 35 patients with the C/C or C/T genotype, whereas lymph node metastasis was found in 37 patients and not found in 16 patients with the T/T genotype (see Table 2 “Relationship between CRP genetic polymorphism and lymph node metastasis”).







TABLE 2







Relationship between CRP genotypes and lymph node metastasis










Lymph node metastasis












Negative
Positive



CRP genotypes
(N = 51)
(N = 62)
P













CRP −717T/C (rs2794521)





genotypes


T/T
35
50


T/C
15
12


C/C
1
0
0.2302


T/T
35
50


T/C + C/C
16
12
0.1891


CRP 1059G/C (rs1800947)


genotypes


G/G
48
58


G/C
3
4
>0.9999


CRP 1444C/T (rs1130864)


genotypes


C/C
49
56


C/T
1
6


T/T
1
0
0.1350


C/C
49
56


C/T + T/T
2
6
0.2906


CRP 1846C/T (rs1205)


genotypes


C/C
6
7


C/T
29
18


T/T
16
37
0.0068*


C/C + C/T
35
25


T/T
16
37
0.0043*





*Significant difference






On the other hand, with regard to the CRP1059G>C (rs1800947) genetic polymorphism, lymph node metastasis was found in 58 patients and not found in 48 patients with the G/G genotype, whereas lymph node metastasis was found in 4 patients and not found in 3 patients with the G/C genotype. Regarding the CRP-717T>C (rs2794521) genetic polymorphism discussed in Non Patent Literature 11 (Motoyama et al., The Japanese Journal of Gastroenterological Surgery vol. 41, No. 7, pp. 1169, July 2008), lymph node metastasis was found in 12 patients and not found in 16 patients with the T/C and C/C genotypes, and regarding the CRP1444C>T (rs1130864) genetic polymorphism, lymph node metastasis was found in 56 patients and not found in 49 patients with the C/C genotype (see Table 2 “Relationship between CRP genetic polymorphism and lymph node metastasis”).
As a result of multivariate logistic analysis using the CRP1846C>T (rs1205) genetic polymorphism and various clinical factors related to lymph node metastasis as covariates, it was revealed that the T/T genotype in a patient at rs1205 is significantly associated with lymph node metastasis (odds ratio ≧3). Conversely, as a result of similar analysis of preoperative serum CRP and SCC levels, tumor size, and age, the odds ratio was approximately 1 in each case. A comparison of the depth of tumor invasion (T2 to 4 versus T1) revealed lymph node metastasis involvement at an odds ratio of 2.571 (see Table 3 “Multivariate logistic regression analysis of lymph node metastasis”).







TABLE 3







Multivariate logistic regression analysis of lymph node metastasis













β
Wald χ2
P
Odds ratio
95% CI
















CRP 1846C/T genotypes (T/T versus C/C + C/T)
1.112
5.615
0.0178
3.040
1.212-7.625


Preoperative serum CRP
−0.107
0.078
0.7799
0.898
0.423-1.907


Serum SCC
0.070
0.252
0.6160
1.072
0.816-1.409


Tumor location (upper versus middle-lower)
0.608
0.297
0.5855
1.836
0.207-16.312


Tumor size
−0.005
0.209
0.6472
0.995
0.975-1.016


Depth of tumor invasion (T2-4 versus T1)
0.944
2.700
0.1004
2.571
0.833-7.929


Tumor differentiation (well-moderately versus poorly)
−0.496
0.710
0.3995
0.609
0.192-1.930


Venous invasion (positive versus negative)
1.131
2.680
0.1016
3.099
0.800-12.003


Intramural metastasis (positive versus negative)
0.219
0.085
0.7705
1.245
0.285-5.439


Age
−0.049
2.246
0.1340
0.952
0.893-1.015


Gender (male versus female)
−0.821
1.278
0.2582
0.440
0.106-1.826





Likelihood-ratio χ2 test, χ2 = 23.241(df = 11), P = 0.0163






With regard to the rs1205 genetic polymorphism, preoperative serum CRP levels were 0-5 mg/L for 43 of the patients with the C/C and C/T genotypes and 43 of the patients with the T/T genotype whereas the levels were greater than 5 mg/L for 16 of the patients with the C/C and C/T genotypes and 8 of the patients with the T/T genotype. (see Table 4 “Relationship between CRP1846C>T (rs1205) genotypes and preoperative serum CRP level, depth of tumor, and number involved lymph nodes”).







TABLE 4







Relationship between CRP 1846C/T (rs1205) genotypes and preoperative serum CRP


level, depth of tumor, and number of involved nodes












CRP 1846C/T (rs1205) genotypes

CRP 1846C/T (rs1205) genotypes
















C/C (N = 13)
C/T (N = 47)
T/T (N = 53)
P
C/C + C/T (N = 60)
T/T (N = 53)
P

















Preoperative serum CRP (mg/L)a
5.7 ± 5.8
4.6 ± 10.6
3.8 ± 6.0
0.737
4.8 ± 9.7
3.8 ± 6.0
0.0537


0-5 mg/L
6
37
43

43
43


>5 mg/L
7
 9
 8
0.0107*
16
 8
0.1706


Depth of tumor invasion (pT)


T1
2
24
15

26
15


T2-4
11 
23
38
0.0153a
34
38
0.1184


Number of
1.4 ± 1.9
1.8 ± 3.7 
2.7 ± 4.9
0.4605
1.7 ± 3.7
2.7 ± 4.9
0.2284


involved lymph nodes


0-2
11
39
35

50
35


>3
2
 8
18
0.1038
10
18
0.0487*





aPreoperative serum CRP level was not measured in one patient in C/T genotype group or in two patients in T/T genotype group






The diagnosability was extremely high after limiting the analysis to subjects with submucosal esophageal cancer (33 patients), in which diagnosis of lymph node metastasis is particularly difficult and approaches to therapy is significantly influenced by the presence of pretreatment lymph node metastasis. Although the lymph node metastasis diagnosis using the most advanced diagnostic imaging apparatus (CT and ultrasonography) achieved a sensitivity, specificity, positive predictive value, and negative predictive value of 50%, 79%, 54%, and 68%, respectively, the diagnosis using the CRP1846C>T (rs1205) genetic polymorphism achieved a sensitivity, specificity, positive predictive value, and negative predictive value of 64%, 79%, 69%, and 75%, respectively, which were more favorable (see Table 5 “Prediction of lymph node involvement in submucosal esophageal cancer using CRP1846C>T (rs1205) genetic polymorphism or the usual methods (CT and ultrasonography)”).







TABLE 5







Prediction of lymph node involvement in submucosal esophageal


cancer using CRP 1846C > T (rs1205) polymorphism


or the usual methods (CT and ultrasonography)














Positive
Negative



Sensi-
Speci-
predictive
predictive



tivity (%)
ficity (%)
value (%)
value (%)















CRP 1846C > T (rs1205)
64
79
69
75


polymorphism


Usual methods
50
79
54
68









Example 2
Similarly, as a result of investigating the relationship between the CRP1846C>T (rs1205) genetic polymorphism and pathological lymph node metastasis in 152 lung cancer patients (all Japanese) who underwent surgery, a significant relationship between the two was revealed as observed for esophageal cancer (Fisher's exact test, P=0.0312).
As indicated by the results above, according to the present invention, lymph node metastasis or the risk thereof can be detected with high accuracy. In particular, lymph node metastasis or the risk thereof can notably be determined with higher accuracy using the CRP1846C>T (rs1205) genetic polymorphism than with polymorphisms of other cytokines involved in CRP production.
Example 3
The relationship between the CRP1846C>T (rs1205) genetic polymorphism and lymph node metastasis was analyzed in 64 patients having a wall invasion depth of pT1-2 among the 113 subjects of Example 1. Regarding the CRP1846C>T (rs1205) genetic polymorphism, lymph node metastasis was found in 6 patients and not found in 35 patients with the C/C or C/T, whereas lymph node metastasis was found in 18 patients and not found in 5 patients with the T/T genotype. The CRP1846C>T (rs1205) genetic polymorphism was significantly related to lymph node metastasis (Fisher's exact test, P=0.0001). As lymph node metastasis can certainly be detected in early cancer, in which lymph node metastasis may be overlooked by conventional diagnostic imaging methods, it was confirmed that the method of the present invention can meet clinical needs.
Example 4
The relationship between the CRP1846C>T (rs1205) genetic polymorphisms and lymphatic vessel invasion was analyzed in 144 patients implicated for lymphatic vessel invasion among the 152 subjects of Example 2. Regarding the CRP1846C>T (rs1205) genetic polymorphism, lymphatic vessel invasion was found in 36 patients and not found in 42 patients with the C/C or C/T genotype, whereas lymphatic vessel invasion was found in 45 patients and not found in 21 patients with the T/T genotype. The CRP1846C>T (rs1205) genetic polymorphism was significantly related to lymphatic vessel invasion (Fisher's exact test, P=0.008). “Lymphatic vessel invasion” indicates the state in which the presence of cancer cells is observed in lymphatic vessel of the primary focus and suggests the possibility of future “lymph node metastasis” even if no “lymph node metastasis” actually exists. Therefore, identifying the genotype of the CRP1846C>T (rs1205) genetic polymorphism is useful in the determination (prediction in earlier stage) of the risk of “lymph node metastasis.”

## Claims

1. A method of treating cancer in a human subject, comprising:
obtaining a biological sample from the subject, said sample comprising nucleic acids from the subject;
detecting a T/T genotype of SNP rs1205 in human C-reactive protein (CRP) gene in the nucleic acids;
correlating the presence of said T/T genotype of SNP rs1205 with an increased risk of lymph node metastasis in the subject; and
administering to the subject a cancer treatment involving lymph node dissection.

2. The method according to claim 1, wherein the genotype is identified by RFLP or its binding with a corresponding complementary strand sequence.

3. The method according to claim 2, wherein the genotype is identified by PCR-RFLP.

4. The method according to claim 3, wherein a forward primer, 5′-CTT ATA GAC CTG GGC (SEQ ID No. 1), and a reverse primer, 5′-GGA GTG AGA CAT CTT CTT G-3′ (SEQ ID No. 2), are used as primers in PCR and Bst4CI is used as a restriction enzyme.

5. The method according to claim 1, wherein the cancer is a solid cancer.

6. The method according to claim 1, wherein the sample is selected from the group consisting of whole blood, leukocytes, the primary focus of cancer, lymphatic vessel, and lymph node tissue.

7. The method according to claim 1, comprising causing a nucleic acid to specifically hybridize with a DNA fragment derived from a region containing SNP rs1205 of the human C-reactive protein (CRP) gene, with the region being amplifiable by a PCR method using primers of SEC), ID Nos. 1 and 2.

8. The method according to claim 5, wherein the solid cancer is selected from the group consisting of: esophagus, lung, breast, head and neck, stomach, colon, biliary tract, pancreas, uterus, ovary, bladder, kidney, urothelium and prostate cancer.

9. The method according to claim 1, wherein the cancer treatment involving lymph node dissection is surgery invoking lymph node dissection.