Patent Application
20070172857
The present invention relates to a primer for a nucleic acid relevant to a colon cancer marker and a method for detecting lymph node metastasis of colon cancer.
In diagnosis of colon cancer, detection of the cancer cell in the lymph node (lymph node metastasis diagnosis) is valuable in determining the operating method or in determining the post-operative chemical therapy. Currently, tissue diagnosis (e.g., HE (hematoxylin-eosin) stain, IHC (immunohistochemistry) method, etc.) using frozen section or paraffin section of the lymph node tissue collected from the living body is mainly performed by pathologists in the lymph node metastasis diagnosis. However, only the cross section of a fragment of the lymph node can be observed with such method. Thus, the cancer cell tends to be overlooked if the cancer cell is present at the fragment of the excised lymph node.
In view of such a situation, research of molecular diagnosis of the cancer using LAMP (loop-mediated isothermal amplification) method, PCR (polymerase chain reaction) method and the like is currently being actively performed. The molecular diagnosis can be performed by detecting the molecule marker (e.g., protein specifically appearing in the cancer cell, gene coding the protein, mRNA of the gene, etc.). For example, cytokeratin 20 (CK 20), and carcino embryonic antigen (CEA) are reported as the molecule marker (hereinafter also referred to simply as marker) for determining the lymph node metastasis of the colon cancer. These molecules are proteins in which a significant difference is recognized between the expression level in the normal lymph node and the expression level in the colon cancer cell metastasized to the lymph node.
The scope of the present invention is defined solely by the appended claims, and is not affected to any degree by the statements within this summary.
A first aspect of the present invention relates to a for amplifying a nucleic acid, comprising any one of nucleotide sequences of SEQ ID NOs: 39 to 106, being capable of hybridizing a cDNA corresponding to a marker for lymph node metastasis of a cancer cell derived from a colon cancer, and being capable of amplifying the cDNA, the marker comprising a mRNA or a fragment of the mRNA, the mRNA being transcribed from a gene coding a protein selected from the group consisting of:
A second aspect of the present invention relates to a method for detecting lymph node metastasis of a cancer cell derived from a colon cancer by detecting a marker in a sample prepared from a lymph node obtained from a living body, wherein the marker is a mRNA or a fragment of the mRNA, the mRNA being transcribed from a gene coding a protein selected from the group consisting of above-stated 34 proteins.
The marker in the present embodiment includes mRNA or a fragment of the mRNA of the gene coding the protein expressed in the cancer cell derived from the colon cancer. The cancer cell in the lymph node can be detected by detecting the marker. In the present specification, “detect” does not only mean determining the presence but also means quantifying. The “mRNA” does not include only the mature mRNA, but also includes the precursor of the mRNA (splicing after transcription, mRNA before polyadenylation, etc.). The marker is the region of the mRNA or a fragment of the mRNA corresponding to Polymeric immunoglobulin receptor (PIGR); Claudin 3 (CLDN3); Galectin 4 (LGALS4); Anterior gradient 2. (AGR2); Tumor-associated calcium signal transducer 1 (TACSTD1); Glutathione peroxidase 2 (GPX2); Retinoic acid induced 3 (RAI3); Tetraspan 1 (TSPAN1); Creatin kinase-brain (CKB); E74-like factor 3 (ELF3); FXYD domain containing ion transport regulator 3 (FXYD3); Cadherin 1 (CDH1); Regenerating islet-derived family member 4 (REG4); Growth differentiation factor 15 (GDF 15); Claudin 4 (CLDN4); Olfactomedin 4 (OLFM 4); CD9 antigen (CD9); Cadherin 17 (CDHl7); Selenium binding protein 1 (SELENBP); Lipocalin 2 (LCN2); Transmembrane protease serin 4 (TMPRSS4); Cystic fibrosis transmembrane conductance regulator ATP binding cassette (CFTR); Transmembrane 4 superfamily member 3 (TM4SF3); Inhibitor of DNA binding 1, dominant negative helix-loop-helix protein (IDl); Cytochrome P450, family 2, subfamily S, polypeptide 1 (CYP2S1); Trefoil factor 3 (TFF3); Ets homologous factor (EHF); FAT tumor suppressor homolog 1 (FAT); Kruppel-like factor 5 (KLF5); Solute carrier family 9 (sodium/hydrogen exchanger), isoform 3 regulator 2 (SLC9A3R2); Homeobox protein B9 (HOXB9); ATPase, Na+/K+ transporting, betal polypeptide (ATP1B1); Phosphoenolpyruvate carboxykinase 1 (PCK1);or Fc fragment of IgG binding protein (FCGBP).
These proteins are expressed in excess in the cancer cell derived from the colon cancer. “Expressed in excess” means being present in great amount in the cancer cell derived from the colon cell compared to the amount present in the normal cell of the lymph node.
A detection sample is preferably prepared first to detect the marker. The detection sample is not particularly limited as long as it is the sample containing polynucleotide that acts as the template of nucleic acid amplification reaction to be hereinafter described, and the sample prepared by solubilizing the cell of the lymph node collected from the living body may be used. The sample including the cell of the lymph node includes cell mass including the cells of the lymph node dissected through operation, sample including the cells of the lymph node collected by biopsy, etc.
The preparation of the detection sample may be performed in the following manner. First, a reagent for solubilization (hereinafter referred to as solubilizing agent) is added to the cell of the lymph node. The cells in the solubilizing agent are crushed with a homogenizer and the like to free the molecules in the cell membrane to the solution. Centrifugal separation is then performed to collect the supernatant, which may then be used as the detection sample. Processes such as nucleic acid purification or nucleic acid extraction may be performed before centrifugal separation and/or after centrifugal separation.
A primer for detecting the marker, RNA dependent DNA polymerase (hereinafter also referred to as reverse transcriptase), and DNA dependent DNA polymerase (hereinafter simply referred to as DNA polymerase) are added to the obtained detection sample to prepare a reaction liquid, and nucleic acid amplification is performed. The nucleic acid amplification method is not particularly limited, and known methods such as PCR method, LAMP method and the like may be used.; The nucleic acid amplification method including reverse transcription reaction before the nucleic acid amplification reaction (e.g., RT-PCR method, RT-LAMP method, etc.) may be used since the marker is an RNA. The CDNA is reverse transcribed from the mRNA of the marker, and such cDNA is further amplified by using the nucleic acid amplification method.
The reverse transcription reaction and the nucleic acid amplification reaction may have the conditions appropriately changed according to the sequence of template cDNA, and the sequence of the primer. The conditions of the reverse transcription reaction and the nucleic acid amplification reaction may be those described in, for example, Sambrook, J. et al. (1989) Molecular Cloning: A Laboratory Manual (2nd ed.), Cold Spring Harbor Laboratory Press, New York.
The primer for detecting the marker is not a particularly limited sequence as long as it is a polynucleotide that can amplify the cDNA corresponding to the marker. The length of the primer is preferably 5 to 50 nucleotides, and more preferably, 10 to 40 nucleotides. The primer may be manufactured through a nucleic acid synthesizing method known in the art.
The above-described primer may include one or more mutations (substitution, deletion, insertion, addition, etc.) of the nucleotide if including a primer function. “Primer function” is a function of hybridizing to the cDNA (cDNA reverse transcribed from the marker.or complementary strand of cDNA) corresponding to the marker to become the start point of the elongation reaction in the nucleic acid amplification. “Hybridize” refers to a phenomenon in which two types of single-stranded polynucleotide forms a double-stranded polynucleotide through hydrogen bonding based on the complementarity of the base under a stringent condition. “Stringent condition” is a condition generally used by those skilled in the art when performing hybridization of the polynucleotide, and is not particularly limited as long as it is a condition in which the primer of the present embodiment can hybridize to the cDNA or the mRNA. The stringency in time of hybridization is known to be a function of temperature, salt concentration, chain length of the primer, GC content of the nucleotide sequence of the primer, and concentration of the chaotropic agent in the hybridization buffer solution. The stringent condition used may, for example, be conditions described in Sambrook, J. et al. (1998) Molecular Cloning: A Laboratory Manual (2nd ed.), Cold Spring Harbor Laboratory Press, New York. Specifically, “hybridization temperature of 42° C. in a solution including 50% of formamide, 5×SSC (150 mM of Nacl, 15 mM of sodium citrate), 50 mM of sodium phosphate, pH 7.6, 5× Denhardt's solution, 10% of dextran sulfuric acid, and 20 μg/ml of nucleic acid” is illustrated, but is not limited thereto.
The polynucleotide-with mutation has complementarity of greater than or equal to 60% preferably, more preferably 80%, and most preferably 95% with respect to a region where the polynucleotide in the cDNA sequence transcribed from the marker hybridizes. In order for such polynucleotide to function as the primer, preferably the 3-base on the 3′ end side of the polynucleotide is completely complementary, and more preferably, the 5-base on the 3′ end side is completely complementary.
The above primer may be used as a primer set made up of a combination of a first primer and a second primer (forward primer and reverse primer) that can amplify the cDNA corresponding to the marker through the nucleic acid amplification method.
The above primer may be modified through a technique normally used in the prior art. The labeling of the primer may be performed using radioactive elements or non-radioactive molecules. The radioactive isotope includes 32p, 33p, 35S, 3H or 125I. The non-radioactive substance is selected from a ligand such as biotin, avidin, streptavidin, or digoxigenin; hapten, pigment and luminescent reagents such as radioluminescent, chemical luminescent, biological luminescent, fluorescent, or phosphorescent reagent.
The reverse transcriptase and the DNA polymerase may be those well known in the art. The reverse transcriptase includes AMV (Avian Myeloblastosis Virus) reverse transcriptase, M-MLV (Molony Murine Leukemia Virus) reverse transcriptase, etc. The DNA polymerase may be Taq DNA polymerase, Pfu DNA polymerase, T4 DNA polymerase, Bst DNA polymerase, etc.
The marker can be detected by detecting the product generated through the nucleic acid amplification described above.
For example, the marker can be detected by detecting the amplified cDNA.
The detection of the amplified cDNA is performed by mixing the reaction solution with a fluorescent intercalator such as ethidium bromide, SYBRGreen, etc. to stain the cDNA in the reaction solution, and measuring the fluorescence intensity of the reaction solution. Furthermore, the marker can be quantified by adding the above fluorescent intercalator to the reaction solution in advance and measuring the fluorescence intensity of the reaction solution in real time.
Furthermore, when magnesium pyrophosphate is produced as the by-product through the amplification of the cDNA, the cDNA can be detected by detecting the same. Since magnesium pyrophosphate is insoluble, the reaction solution becomes clouded with increase in the magnesium pyrophosphate. Therefore, the cDNA can be detected by optically measuring (e.g., turbidity measurement, absorbance measurement, etc.) the reaction solution. Furthermore, the marker can be quantified by performing optical measurement in real time.
The markers of the present embodiment include not only those in the cancer cells but also those recognized by a small amount in normal cells. Preferably, in this case, the lymph node metastasis is detected by quantifying the marker, and comparing the result with a predetermined threshold.
For example, when quantifying the marker in real time using RT-PCR, the lymph node metastasis can be detected by comparing the number of PCR cycles until reaching a predetermined fluorescence intensity or turbidity with the corresponding threshold value. Furthermore, the lymph node metastasis can be detected by comparing the fluorescence intensity or the turbidity in the predetermined number of cycles with the corresponding threshold value.
For example, when quantifying the marker in real time using RT-LAMP, the lymph node metastasis can be detected by comparing the time until reaching a predetermined fluorescence intensity or turbidity with the corresponding threshold value. Furthermore, the lymph node metastasis can be detected by comparing the fluorescence intensity or the turbidity at the point a predetermined time has elapsed with the corresponding threshold value. Moreover, the lymph node metastasis of the cancer cell can be detected in multi-stages such as “most positive”, “positive”, “negative”, etc. by setting a plurality of threshold values.
The threshold value is set to a value lower than or equal to the marker amount contained in the living body sample that has been confirmed that cancer cells are included (positive sample), and higher than the marker amount contained in the living body sample that has been confirmed that cancer cells are not included (negative sample). The threshold value is preferably a value. for measuring the marker amount of a plurality of positive samples and the marker amount of a plurality of negative samples, and distinguishing the positive sample and the negative sample at the highest probability.
A micro array technique may also be used to detect the marker. Specifically, the polynucleotide probe (hereinafter simply referred to as probe) complementary to the cDNA corresponding to the marker is first immobilized in the solid phase. The sample containing the cDNA is added to the relevant solid phase to capture the cDNA by the probe. The phosphorescence intercalator is added thereafter to phosphorescent stain the hybrid of the probe and the cDNA, and then the phosphorescence intensity is detected. The quantification of and the determination of the presence of the marker is performed from the detection result of the phosphorescence intensity. If the probe is shorter than the cDNA, the second probe that hybridizes to the region of the cDNA where the probe is not hybridized may be added to intensify the phosphorescence signal.
The probe may be designed and manufactured through a method similar to that described for the primer. A probe having a sequence similar to the primer may be used for the relevant probe.
The reagent or the like necessary to detect the marker of the present embodiment is provided as a reagent kit. The kit includes at least the above-described primer, enzyme having reverse transcript activity, DNA polymerase and dNTPs (DATP, dGTP, dTTP, dCTP). This kit preferably includes a buffer agent that provides a condition suitable for the enzyme reaction.
In the present specification, “detecting the marker” not only means detecting the entire region of the mRNA acting as the marker, but also means detecting a partial region. Preferably, the cDNA corresponding to one fragment of the region of the marker is amplified, and then detected in the present embodiment. In this case, the length of the region of the detected cDNA is longer than the length of the primer preferably by 1 to 500 nucleotides, and more preferably by 50 to 500 nucleotides. Furthermore, when using the primer set, the length of the region of the amplified cDNA is preferably longer than the sum of the length of the first primer and the length of the second primer by 1 to 500 nucleotides, and more preferably by 50 to 500 nucleotides.
The inventors searched for a marker with which metastasis to the lymph node of the colon cancer can be detected. The gene expression library related to the colon was selected from the human gene expression library registered in a public database, and 98 types of genes which representation at the colon is high and representation at the lymph node is low were selected from the database from the top in the order of high representation at the colon. The proteins corresponding to such genes are shown in Table 1 below. Next, 98 sets of primer with which the mRNA (98 types of mRNA are hereinafter referred to as marker candidates) of the gene coding the 98 types of protein can be detected were designed.
The positive sample and the negative sample were prepared in the following manner using four lymph nodes (positive lymph nodes) in which metastasis of the cancer cells deviated from the colon cancer was histologically recognized, and four lymph nodes (negative lymph nodes) in which metastasis of the cancer cells deviated from the colon cancer was not histologically recognized.
Four mL of solubilizing solution (containing 200 mM glycin-HCl, 5% of Brij 35 (polyoxyethylene (35) laurel ether), 20% of DMSO, and 0.05% of KS-538 (Shin-Etsu Chemical Co., Ltd.)) was added to each lymph node (about 100-300 mg/node) and the lymph node was homogenized with a blender in the solution. The obtained homogenate was performed with centrifugal separation for one minute at 10000xg and at room temperature, RNA was extracted and purified from 400 μL of supernatant using RNeasy Mini kit (manufactured by QIAGEN K.K., catalog number 74014) and the RNA solution was obtained. After measuring the absorbance (λ=280 nm) of the RNA solution and checking the concentration, the solution was diluted to 10 ng/μL RNA concentration. Five solutions each obtained form 5 positive lymph nodes were mixed to prepare “positive analyte.” And 5 solutions each obtained from 5 negative lymph nodes were mixed to prepare “negative analyte.”
The sample in which the obtained RNA solution was mixed for five positive analytes (hereinafter referred to as “positive analyte”) and the sample in which the obtained RNA solution was mixed for five negative analytes (hereinafter referred to as “negative analyte”) were obtained.
The positive analyte and the negative analyte obtained as above were performed with real-time RT-PCR by means of the ABI real-time PCR device (Prism 7000) using 98 sets of primers, and detection of 98 types of mRNA was performed.
The real-time RT-PCR was performed using the Quanti Tect SYBR Green RT-PCR kit (manufactured by QIAGEN K.K., catalog number 204245), a quantifying RT-PCR kit, according to the manual. The composition and the reaction conditions of the reaction solution are as described below.
Reaction Solution:
The RT-PCR was performed in the above conditions. The number of negative analyte PCR cycle (the number of PCR cycles when fluorescence of reaction solution including negative analyte was reached a certain specific fluorescence intensity) and the number of positive PCR cycle (the number of PCR cycles when fluorescence of reaction solution including positive analyte was reached a certain specific fluorescence intensity) were measured. The difference of (number of negative analyte PCR cycle)−(number of positive analyte PCR cycle) was calculated. The larger the difference in the number of PCR cycles, the less the representation of the gene in the negative analyte, and the more the representation in the positive analyte, that is, the gene has specifically more representation at the lymph node where metastasis has occurred.
This result is shown in Table 1 below and
Therefore, the mark candidates having a large difference in the number of PCR cycles are effective as a marker for detecting metastasis to the lymph node of the colon cancer.
The β-actin (ACTB) that was used as a control is known as a protein of the housekeeping gene and is expressed in large amount in many cells. Therefore, the difference between the number of cycles in the negative RNA sample and the number of cycles in the postive RNA sample is not found.
Furthermore, the mRNA of the CK 20 and the CEA that have been known as the lymph node metastasis marker from the prior art have a relatively large difference in the number of PCR cycles, and specifically have a great amount of expression in the tissue where lymph node metastasis occurred.
From the above results, the mRNA of the gene coding 34 types of porteins shown in Table 2 below are newly identified as the lymph node metastasis marker. In Table 2, the Seq ID Nos of the mRNA of the genes each coding the respective 34 types of proteins is shown in the column of “Seq ID No”. The sequence of the mRNA is as shown in sequence numbers 1 to 38. The sequence of the Seq ID Nos 1 to 38 is represented by substituting U (uracil) in the mRNA sequence with T (thymine). These sequences are available with the following accession number from Genbank (http://www.ncbi.nlm.nih.gov/Genbank/index.html) in table 2. “Lymph node metastasis marker” in the present specification is a polynucleotide having the entire length sequence of the mRNA shown in sequence numbers 1 to 38 or a polynucleotide having a partial sequence thereof.
The primers used in detecting the above markers and the corresponding Seq ID NOs are shown in Table 3 below. The primers are written in the direction of 5′→3′ from left to right.
The real-time RT-PCR was respectively performed using the RNA solution extracted from six positive lymph nodes (lymph node metastasis was histologically recognized) each collected from six patients (analyte numbers 8, 10, 21, 29, 37 and 59: positive sample); and the RNA solution extracted from twenty-four negative lymph nodes (lymph node metastasis was not histologically recognized) each collected from twenty-four patients (analyte numbers 1, 3, 4, 5, 6, 12, 13, 15, 16, 18, 19, 20, 22, 23, 24, 26, 27, 28, 29, 30, 32, 34, 35 and 38: negative sample). Twelve types of marker (PIGR, CLDN3, LGALS4, AGR2, TSPAN-1, FXYD3, CLDN4, OLFM4, CDH17, LCN2, TMPRSS4, and CFTR), mRNA of CK 20, and mRNA of ACTB shown in Table 4 below were detected. The method of preparing the RNA solution and the conditions of the RT-PCR are similar to example 1. In addition, RT-PCR was performed similar to above with the reaction solution mixed with purified water as negative control (NTC) in place of the analyte containing RNA. The above experiments were performed twice. The primers used in RT-PCR are as shown in Table 4 below.
In the real-time RT-PCR, the result of measuring the number of PCR cycles (Ct) until reaching a specific fluorescence intensity is shown in FIGS. 3 to 16.
In the detection experiment of the mRNA of the ACTB, the number of cycles necessary for the cDNA transcribed from the mRNA to be amplified to a certain amount in all the analytes other than NTC is few, and thus a great amount of mRNA is detected.
In the detection experiment of the mRNA of the CK 20 and the twelve types of markers, the number of cycles required in the amplification is small in the sample where the metastasis to the lymph node is histologically recognized, whereas the number of cycles required in the amplification is large in the sample where the metastasis to the lymph node is not recognized. Therefore, the lymph node metastasis markerscan be suitably used in detecting the metastasis to the lymph node of the cancer cell.
According to the above results, all the positive samples used in the present example are determined as positive, and all the negative samples are determined as negative by setting the number of cycles of 29 as the threshold value in the detection of the mRNA of the PIGR.
All the positive samples used in the present example are determined as positive, and all the negative samples are determined as negative by setting the threshold value in a range of number of cycles of 24 to 26 in the detection of the mRNA of CLDN3.
The positive samples out of the positive samples used in the present example are determined as positive at high probability, and all the negative samples are determined as negative by setting the number of cycles of 26 as the threshold in the detection of the mRNA of LGALS4.
All the positive samples used in the present example are determined as positive, and all the negative samples are determined as negative by setting the threshold value in a range of number of cycles of 25 to 28 in the detection of the mRNA of AGR2.
All the positive samples used in the present example are determined as positive, and all the negative samples are determined as negative by setting the threshold value in a range of number of cycles of 30 to 31 in the detection of the mRNA of TSPAN-1.
All the positive samples used in the present example are determined as positive, and all the negative samples are determined as negative by setting the threshold value in a range of number of cycles of 25 to 27 in the detection of the mRNA of FXYD3.
All the positive samples used in the present example are determined as positive, and all the negative samples are determined as negative by setting the threshold value in a range of number of cycles of 25 to 26 in the detection of the mRNA of CLDN4.
The positive samples out of the positive samples used in the present example are determined as positive at high probability, and the negative samples are determined as negative at high probability by setting the number of cycles of 32 as the threshold in the detection of the mRNA of OLFM4.
All the positive samples used in the present example are determined as positive, and all the negative samples are determined as negative by setting the threshold value in a range of number of cycles of 27 to 28 in the detection of the mRNA of CDH17.
All the positive samples used in the present example are determined as positive, and all the negative samples are determined as negative by setting the threshold value in a range of number of cycles of 28 to 30 in the detection of the mRNA of LCN2.
All the positive samples used in the present example are determined as positive, and all the negative samples are determined as negative by setting the threshold value in a range of number of cycles of 28 to 30 in the detection of the mRNA of TMPRSS4.
All the positive samples used in the present example are determined as positive, and the negative samples are determined as negative at high probability by setting the threshold value in a range of number of cycles of 27 to 30 in the detection of the mRNA of CFTR.
When the sample prepared from the lymph node at where the presence of the lymph node metastasis of the cancer cells is unknown is used, whether or not the cancer cells deviated from the colon cancer has metastasized to said lymph node can be detected by comparing the threshold value and the number of cycles measured using the relevant sample.
The lymph node metastasis detection of higher accuracy may be performed by combining two or more detection results of the twelve types of markers.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2005-379718 | Dec 2005 | JP | national |
