Method, polynucleotide, and kit for evaluating the risk of developing gastric cancer in patients after Helicobacter pylori eradication.

Abstract

Provided is a method for assessing the risk of the onset of stomach cancer in a patient after removing Helicobacter pylori, the method comprising a step for detecting the expression of a KK-LC-1 gene in a stomach tissue sample derived from the patient before removing Helicobacter pylori, wherein positive expression of the KK-LC-1 gene indicates that there is a risk of the onset of stomach cancer in the patient after removing Helicobacter pylori.

Description

Technical Field

[0001] The present invention relates to a method for evaluating the risk of a patient developing gastric cancer after Helicobacter pylori eradication, a polynucleotide, and a kit. This application claims priority based on Japanese Patent Application No. 2022-140097 filed in Japan on September 2, 2022, the content of which is incorporated herein by reference.

Background Art

[0002] It is known that Helicobacter pylori infection is one of the causes of gastric cancer development. Currently, when Helicobacter pylori infection is detected, eradication by oral administration is covered by insurance. It has been reported that the risk of gastric cancer development in cases where Helicobacter pylori has been eradicated is one-third compared to the non-eradicated group. On the other hand, this result indicates that there are cases where cancer develops even after Helicobacter pylori eradication. At present, there is no technology for evaluating the risk of developing gastric cancer after Helicobacter pylori eradication. That is, even if eradication is successful, the target patients remain anxious about developing cancer as before. Furthermore, since there is no cancer development risk index after eradication, regular check-ups are required for all cases of successful eradication, and neither medical economy nor medical resources are reduced. The risk index as described above is an unmet medical need strongly demanded from both the medical field and the patient side.

[0003] By the way, cancer / testis antigen is a general term for proteins that are not expressed outside cancer cells and the testis. Kitakyushu lung cancer antigen-1 (KK-LC-1) is one type of cancer / testis antigen. The inventors have hitherto clarified that KK-LC-1 can be used as a marker expressed in various cancers (see, for example, Patent Document 1).

Prior Art Documents

Patent Documents

[0004]

Patent Document 1

[0005] The present invention aims to provide a technology for evaluating the risk of developing gastric cancer in patients after eradication of Helicobacter pylori. [Means for solving the problem]

[0006] The present invention includes the following embodiments. [1] A method for evaluating the risk of a patient developing gastric cancer after eradication of Helicobacter pylori, comprising the step of detecting the expression of the KK-LC-1 gene in a gastric tissue sample derived from the patient prior to eradication of Helicobacter pylori, wherein a positive expression of the KK-LC-1 gene indicates that the patient is at risk of developing gastric cancer after eradication of Helicobacter pylori. [2] The method according to [1], wherein the expression of the KK-LC-1 gene is detected by endpoint RT-PCR. [3] The method according to [2], wherein in the endpoint RT-PCR, a primer consisting of the nucleotide sequence described in SEQ ID NO: 1 and a primer consisting of the nucleotide sequence described in SEQ ID NO: 2 are used. [4] The method according to [1], wherein the expression of the KK-LC-1 gene is detected by real-time RT-PCR. [5] The method according to [4], wherein a qPCR probe is used in the real-time RT-PCR, and the qPCR probe has a nucleotide sequence that includes the 195th and 196th nucleotides in the nucleotide sequence described in Sequence ID No. 3, has a nucleotide length of 15 to 50 nucleotides, and has one or two nucleotide mutations from the nucleotide sequence described in Sequence ID No. 3. [6] The method according to [4] or [5], wherein the real-time RT-PCR is performed in the presence of a single-stranded nucleic acid fragment that suppresses the amplification of genomic DNA, and the single-stranded nucleic acid fragment has a base sequence that includes the 322nd and 323rd bases or the 1096th and 1097th bases in the base sequence described in Sequence ID No. 4, and has a base length of 20 to 50 bases. [7] The method according to [1], wherein the expression of the KK-LC-1 gene is detected at the protein level. [8] A kit for evaluating the risk of developing gastric cancer in patients after eradication of Helicobacter pylori, comprising a primer having the nucleotide sequence described in SEQ ID NO: 1 and a primer having the nucleotide sequence described in SEQ ID NO: 2. [9] The kit according to [8], further comprising a qPCR probe, wherein the qPCR probe has a nucleotide sequence containing the 195th and 196th nucleotides in the nucleotide sequence described in SEQ ID NO: 3, has a nucleotide length of 15 to 50 nucleotides, and has one or two nucleotide mutations from the nucleotide sequence described in SEQ ID NO: 3.

[10] The kit according to [8] or [9], further comprising a single-stranded nucleic acid fragment that suppresses amplification of genomic DNA, wherein the single-stranded nucleic acid fragment has a base sequence that includes the 322nd and 323rd bases or the 1096th and 1097th bases in the base sequence described in Sequence ID No. 4, and has a base length of 20 to 50 bases.

[11] A kit for evaluating the risk of developing gastric cancer in patients after eradication of Helicobacter pylori, comprising a specific binding agent for the KK-LC-1 protein.

[12] A polynucleotide having a nucleotide sequence that includes the 195th and 196th nucleotides in the nucleotide sequence described in Sequence ID No. 3, with a nucleotide length of 15 to 50 nucleotides, and having one or two nucleotide mutations from the nucleotide sequence described in Sequence ID No. 3.

[13] A polynucleotide having a base sequence that includes the 322nd and 323rd bases, or the 1096th and 1097th bases, in the base sequence described in Sequence ID No. 4, and having a base length of 20 to 50 bases.

[14] A method for detecting the expression of the KK-LC-1 gene in a biological sample, wherein the detection is performed by real-time RT-PCR, the real-time RT-PCR is performed using a qPCR probe, the real-time RT-PCR is performed in the presence of a single-stranded nucleic acid fragment that suppresses the amplification of genomic DNA, and the single-stranded nucleic acid fragment has a base sequence that includes the 322nd and 323rd bases or the 1096th and 1097th bases in the base sequence described in Sequence ID No. 4, and has a base length of 20 to 50 bases.

[15] The method according to

[14] , wherein the qPCR probe has a nucleotide sequence that includes the 195th and 196th nucleotides in the nucleotide sequence described in SEQ ID NO: 3, has a nucleotide length of 15 to 50 nucleotides, and has one or two nucleotide mutations from the nucleotide sequence described in SEQ ID NO: 3. [Effects of the Invention]

[0007] The present invention provides a technology for evaluating the risk of developing gastric cancer in patients after eradication of Helicobacter pylori. [Brief explanation of the drawing]

[0008] [Figure 1] Figure 1 is a graph showing the 5-year cumulative incidence rate of gastric cancer in patients in the KK-LC-1 positive and negative groups in Experimental Example 1. [Figure 2] Figure 2 is a graph showing the 5-year cumulative incidence rate of gastric cancer in patients in the KK-LC-1 positive and negative groups in Experimental Example 2. [Figure 3] Figure 3 is a graph showing the results of real-time PCR in Experiment Example 3. [Figure 4] Figure 4 is a graph showing the results of real-time PCR in Experiment Example 3. [Figure 5] Figure 5 is a graph showing the results of real-time PCR in Experiment Example 3. [Figure 6] Figure 6 is a graph showing the results of real-time PCR in Experiment Example 3. [Figure 7]FIG. 7 is a graph showing the results of real-time PCR in Experimental Example 4. [Figure 8] FIG. 8 is a graph showing the results of real-time PCR in Experimental Example 4. [Figure 9] FIG. 9 is a graph showing the results of real-time PCR in Experimental Example 5. [Figure 10] FIG. 10 is a graph showing the results of real-time PCR in Experimental Example 6. [Figure 11] FIG. 11 is a graph showing the results of real-time PCR in Experimental Example 6.

MODE FOR CARRYING OUT THE INVENTION

[0009] [Method for Evaluating the Risk of Gastric Cancer Development in Patients after Helicobacter pylori Eradication] In one embodiment, the present invention provides a method for evaluating the risk of gastric cancer development in a patient after Helicobacter pylori eradication, the method including a step of detecting the expression of the KK-LC-1 gene in a gastric tissue sample derived from the patient before Helicobacter pylori eradication, wherein a positive expression of the KK-LC-1 gene indicates that the patient has a risk of developing gastric cancer after Helicobacter pylori eradication.

[0010] The inventors have found that when the expression of the KK-LC-1 gene is positive in a gastric tissue sample derived from a patient before Helicobacter pylori eradication, the patient has a risk of developing gastric cancer after Helicobacter pylori eradication, and thus completed the present invention. The NCBI accession number of the cDNA of the human KK-LC-1 gene is NM_001017978.4. Also, the NCBI accession number of the genomic DNA of the human KK-LC-1 gene is NC_000023.11. Also, the NCBI accession number of the human KK-LC-1 protein is NP_001017978.1.

[0011] As will be described later in the examples, by the method of the present embodiment, it is possible to evaluate the risk that a patient develops gastric cancer after Helicobacter pylori eradication. Conventionally, there has been no technique for evaluating the risk of developing gastric cancer after Helicobacter pylori eradication.

[0012] As the gastric tissue sample, a gastric tissue sample collected at the time of confirming infection with Helicobacter pylori can be preferably used. When the expression of the KK-LC-1 gene is positive, it indicates that even after Helicobacter pylori eradication, the patient has a risk of developing gastric cancer. In this case, measures such as increasing the examination frequency of gastric cancer in the patient can be taken.

[0013] When the expression of the KK-LC-1 gene is negative, it indicates that the risk of a patient developing gastric cancer after Helicobacter pylori eradication is low. In this case, the examination frequency of gastric cancer in the patient can be reduced, and limited medical resources can be saved. In addition, it is possible to relieve the anxiety of patients who are worried about the development of gastric cancer and reduce the psychological burden of the patients.

[0014] In the method of the present embodiment, the detection of the expression of the KK-LC-1 gene may be performed by endpoint RT-PCR. In this case, it is preferable to use a primer consisting of the nucleotide sequence set forth in SEQ ID NO: 1 and a primer consisting of the nucleotide sequence set forth in SEQ ID NO: 2. Here, endpoint RT-PCR means ordinary RT-PCR, not real-time RT-PCR.

[0015] In the method of the present embodiment, the detection of the expression of the KK-LC-1 gene may be performed by real-time RT-PCR. As will be described later in the examples, by performing the detection of the expression of the KK-LC-1 gene by real-time RT-PCR, the risk that a patient develops gastric cancer after Helicobacter pylori eradication can be evaluated more accurately as compared with endpoint RT-PCR.

[0016] In real-time RT-PCR, it is preferable to use qPCR probes. qPCR probes, also known as TaqMan® probes, are oligonucleotides modified with a fluorescent substance and a quenching substance. qPCR probes specifically hybridize to template DNA during the annealing step in the PCR reaction. Because a quenching substance is present on the qPCR probe, fluorescence generation is suppressed even when irradiated with excitation light.

[0017] In the subsequent extension reaction step, the qPCR probe hybridized to the template is degraded by the 5'→3' exonuclease activity of TaqDNA polymerase. As a result, the fluorescent substance and quenching substance are released, and the inhibition by the quenching substance is released, causing fluorescence to be emitted upon irradiation with excitation light.

[0018] In real-time RT-PCR using qPCR probes, nucleic acid quantification is performed by utilizing the linearity between the PCR amplification product (i.e., the number of cycles at which the fluorescence intensity reaches a certain level) and the initial amount of target DNA.

[0019] In real-time RT-PCR, for example, the difference between the Ct value of β-actin and the Ct value of the KK-LC-1 gene may be calculated and defined as ΔCt. In this case, for example, a ΔCt of -2 or higher may be classified as a high-expression group, a ΔCt of -5 or higher but less than -2 as a medium-expression group, and a ΔCt of less than -5 as a low-expression group.

[0020] The NCBI accession number for the human β-actin gene cDNA is NM_001101.5. The NCBI accession number for the human β-actin protein is NP_001092.1.

[0021] As described later in the examples, the 5-year cumulative incidence of gastric cancer was 40% in the KK-LC-1 high-expression group and 5% in the moderate-expression group. In contrast, the 5-year cumulative incidence of gastric cancer was 0% in the KK-LC-1 low-expression group. Therefore, it can be said that the smaller the ΔCt value, when based on the β-actin Ct value, the lower the risk of the patient developing gastric cancer after Helicobacter pylori eradication.

[0022] In the method of this embodiment, the qPCR probe preferably has a nucleotide sequence that includes the 195th and 196th nucleotides in the nucleotide sequence described in Sequence ID No. 3, has a nucleotide length of 15 to 50 nucleotides, and has a mutation of 1 or 2 nucleotides from the nucleotide sequence described in Sequence ID No. 3.

[0023] The genomic DNA encoding KK-LC-1 contains an exon 1, an intron, and an exon 2. The base sequence of the KK-LC-1 genomic DNA is shown in Sequence ID No. 4.

[0024] The nucleotide sequence described in Sequence ID No. 3 is the nucleotide sequence of the cDNA of the KK-LC-1 gene. The 195th nucleotide in Sequence ID No. 3 corresponds to the last nucleotide of the first exon of KK-LC-1. Also, the 196th nucleotide in Sequence ID No. 3 corresponds to the first nucleotide of the second exon of KK-LC-1.

[0025] In the method of this embodiment, the qPCR probe preferably has a nucleotide sequence that includes the 195th and 196th nucleotides in the nucleotide sequence described in Sequence ID No. 3. That is, the qPCR probe preferably hybridizes to the junction site between the first and second exons in the cDNA of the KK-LC-1 gene.

[0026] Such qPCR probes maintain hybridization ability to the KK-LC-1 gene cDNA while reducing hybridization to the KK-LC-1 gene genomic DNA. As a result, it becomes possible to detect the KK-LC-1 gene cDNA with greater accuracy.

[0027] Furthermore, the base length of the qPCR probe is not particularly limited as long as the Tm value of the qPCR probe can be set appropriately, and may be around 15 to 50 bases, for example, 15 to 40 bases, or for example, around 15 to 30 bases.

[0028] qPCR probes may be modified with a minor groove binder (MGB). Modification with MGB can increase the Tm value. Therefore, modifying qPCR probes with MGB allows for the design of shorter nucleotide lengths.

[0029] MGBs are not particularly limited as long as they increase Tm values, and examples include dihydropyrroloindole tripeptide (CDPI3) and Hoechst33258.

[0030] Furthermore, in the method of this embodiment, it is preferable that the qPCR probe has one or two base mutations in the base sequence (SEQ ID NO: 3) of the cDNA of the KK-LC-1 gene.

[0031] There are similarities between the base sequences of the introns and the second exon of the KK-LC-1 gene. Therefore, qPCR probes that hybridize to the junction site between the first and second exons in the cDNA of the KK-LC-1 gene may also hybridize to the genomic DNA of the KK-LC-1 gene.

[0032] In contrast, as will be described later in the examples, if the qPCR probe has one or two base mutations in the base sequence of the KK-LC-1 gene cDNA (SEQ ID NO: 3), the hybridization ability to the KK-LC-1 gene cDNA is maintained while hybridization of the KK-LC-1 gene to the genomic DNA is reduced. As a result, it becomes possible to detect the KK-LC-1 gene cDNA more accurately. Examples of such qPCR probes include qPCR probes having the base sequence described in any of SEQ ID NOs: 5 to 10. Among these, qPCR probes having the base sequence described in any of SEQ ID NOs: 7 to 10 are preferred.

[0033] In the method of this embodiment, the above-mentioned real-time RT-PCR can also be performed in the presence of a single-stranded nucleic acid fragment that suppresses the amplification of genomic DNA. The above-mentioned single-stranded nucleic acid fragment has a base sequence that includes the 322nd and 323rd bases, or the 1096th and 1097th bases, in the base sequence of the genomic DNA of KK-LC-1 (SEQ ID NO: 4), and preferably has a base length of 20 to 50 bases. The base length of the single-stranded nucleic acid fragment is not particularly limited as long as the Tm value of the single-stranded nucleic acid fragment can be appropriately set, and may be about 20 to 50 bases, for example 20 to 40 bases, for example 25 to 35 bases, for example 20 to 34 bases, or for example 20 to 31 bases.

[0034] The 1096th base in SEQ ID NO: 4 corresponds to the first base of the intron of KK-LC-1. Furthermore, the 1097th base in SEQ ID NO: 4 corresponds to the last base of the first exon of KK-LC-1.

[0035] Therefore, a single-stranded nucleic acid fragment having a base sequence containing the 1096th and 1097th bases in SEQ ID NO: 4 hybridizes to the junction site between the first exon and intron of the KK-LC-1 genomic DNA, thereby suppressing the hybridization of the qPCR probe to the KK-LC-1 genomic DNA. As a result, it becomes possible to detect the cDNA of the KK-LC-1 gene with greater accuracy.

[0036] The 322nd base in Sequence ID No. 4 corresponds to the first base of the second exon of KK-LC-1. Furthermore, the 323rd base in Sequence ID No. 4 corresponds to the last base of the intron of KK-LC-1.

[0037] Therefore, a single-stranded nucleic acid fragment having a nucleotide sequence containing the 322nd and 323rd bases in SEQ ID NO: 4 hybridizes to the intron-exon junction site of the KK-LC-1 genomic DNA, thereby suppressing the hybridization of the qPCR probe to the KK-LC-1 genomic DNA. As a result, it becomes possible to detect the cDNA of the KK-LC-1 gene more accurately. Examples of such single-stranded nucleic acid fragments include single-stranded nucleic acid fragments having the nucleotide sequences described in any of SEQ ID NOs: 11 to 13.

[0038] In the method of this embodiment, the expression of the KK-LC-1 gene may be detected at the protein level. In this case, the KK-LC-1 protein in the gastric tissue sample can be detected by ELISA, Western blotting, or the like using a specific binding agent for KK-LC-1. The specific binding agent will be described later.

[0039] [A kit to assess the risk of developing gastric cancer in patients after Helicobacter pylori eradication] In one embodiment, the present invention provides a kit for evaluating the risk of developing gastric cancer in patients after eradication of Helicobacter pylori, comprising a primer consisting of the nucleotide sequence described in SEQ ID NO: 1 and a primer consisting of the nucleotide sequence described in SEQ ID NO: 2.

[0040] The kit of this embodiment further comprises a qPCR probe having a nucleotide sequence that includes the 195th and 196th nucleotides in the nucleotide sequence described in Sequence ID No. 3, has a nucleotide length of 15 to 50 nucleotides, and may have one or two nucleotide mutations from the nucleotide sequence described in Sequence ID No. 3. The qPCR probe is the same as described above.

[0041] The kit of this embodiment further comprises a single-stranded nucleic acid fragment that suppresses the amplification of genomic DNA, wherein the single-stranded nucleic acid fragment has a base sequence that includes the 322nd and 323rd bases, or the 1096th and 1097th bases, in the base sequence described in Sequence ID No. 4, and may have a base length of 20 to 50 bases. The single-stranded nucleic acid fragment that suppresses the amplification of genomic DNA is the same as described above.

[0042] The kit of this embodiment may include a specific binding agent for the KK-LC-1 protein. Examples of specific binding agents for the KK-LC-1 protein include antibodies against KK-LC-1 and aptamers against KK-LC-1. The antibody may be an antibody fragment. Examples of antibody fragments include F(ab')2, Fab', Fab, Fv, and scFv. Examples of aptamers include nucleic acid aptamers and peptide aptamers.

[0043] These kits can be used to suitably implement the methods described above for evaluating the risk of developing gastric cancer.

[0044] [Polynucleotides] In one embodiment, the present invention provides a polynucleotide having a nucleotide sequence that includes the 195th and 196th nucleotides in the nucleotide sequence described in Sequence ID No. 3, having a nucleotide length of 15 to 50 nucleotides, and having a mutation of 1 or 2 nucleotides from the nucleotide sequence described in Sequence ID No. 3.

[0045] In this embodiment, the polynucleotide is preferably modified with a fluorescent substance and a quenching substance to form a qPCR probe.

[0046] As for the fluorescent substance, any substance commonly used in qPCR probes can be used without any particular restrictions. Specific examples of fluorescent substances include fluorescein, Alexa488, ATTO542, Alexa647, FAM, Cy5, Cy3, etc.

[0047] As for the quenching agent, any substance commonly used in qPCR probes can be used without particular restriction. Specific examples of quenching agents include Black Hole Quencher (BHQ)(registered trademark)-1, BHQ(registered trademark)-2, BHQ(registered trademark)-3, Iowa Black FQ, Iowa Black RQ, etc. The quenching agent should be selected to quench the fluorescence of the fluorescent substance being used.

[0048] The polynucleotide of this embodiment can be suitably used as the qPCR probe described above. By using the polynucleotide of this embodiment as a qPCR probe and performing real-time RT-PCR, it becomes possible to detect the cDNA of the KK-LC-1 gene more accurately.

[0049] In another embodiment, the present invention provides a polynucleotide having a nucleotide sequence including the 322nd and 323rd bases, or the 1096th and 1097th bases, in the nucleotide sequence described in Sequence ID No. 4, and having a nucleotide length of 20 to 50 nucleotides. The polynucleotide of this embodiment can be suitably used as a single-stranded nucleic acid fragment that suppresses the amplification of genomic DNA as described above.

[0050] More specific examples of single-stranded nucleic acid fragments include, for example, single-stranded nucleic acid fragments having the base sequence described in any of sequence numbers 11 to 13. As described above, by performing real-time RT-PCR in the presence of the single-stranded nucleic acid fragment of this embodiment, it becomes possible to detect the cDNA of the KK-LC-1 gene more accurately.

[0051] [Method for detecting KK-LC-1 gene expression in biological samples] In one embodiment, the present invention provides a method for detecting the expression of the KK-LC-1 gene in a biological sample, wherein the detection is performed by real-time RT-PCR, the real-time RT-PCR is performed using a qPCR probe, the real-time RT-PCR is performed in the presence of a single-stranded nucleic acid fragment that suppresses the amplification of genomic DNA, and the single-stranded nucleic acid fragment has a base sequence containing the 322nd and 323rd bases, or the 1096th and 1097th bases, in the base sequence described in Sequence ID No. 4, and has a base length of 20 to 50 bases. The single-stranded nucleic acid fragment is the same as described above.

[0052] As described above, the single-stranded nucleic acid fragment hybridizes to the junction site of the first exon and intron of the KK-LC-1 genomic DNA, thereby suppressing the hybridization of the qPCR probe to the KK-LC-1 genomic DNA. As a result, as will be described later in the examples, it becomes possible to detect the cDNA of the KK-LC-1 gene more accurately. Here, more accurate detection means that false positive detection is suppressed compared to when the single-stranded nucleic acid fragment described above is not used.

[0053] In the method of this embodiment, examples of biological samples include tissues, serum, plasma, cultured cells, and cell culture supernatants derived from humans or non-human animals.

[0054] In the method of this embodiment, it is preferable that the qPCR probe has a nucleotide sequence containing the 195th and 196th nucleotides in the nucleotide sequence described in SEQ ID NO: 3, has a nucleotide length of 15 to 50 nucleotides, and has one or two nucleotide mutations from the nucleotide sequence described in SEQ ID NO: 3. As described above, such a qPCR probe maintains the ability to hybridize to the cDNA of the KK-LC-1 gene while reducing hybridization of the KK-LC-1 gene to the genomic DNA. As a result, it becomes possible to detect the cDNA of the KK-LC-1 gene more accurately. As described above, examples of such qPCR probes include qPCR probes having the nucleotide sequence described in any of SEQ ID NOs: 5 to 10. Among these, qPCR probes having the nucleotide sequence described in any of SEQ ID NOs: 7 to 10 are preferred. [Examples]

[0055] The present invention will now be described in more detail with reference to examples, but the present invention is not limited to the following examples.

[0056] [Experimental Example 1] (Evaluation using endpoint RT-PCR) In 111 patients who underwent eradication therapy for Helicobacter pylori, gastric tissue samples collected before eradication were subjected to RT-PCR to detect the expression of the KK-LC-1 gene.

[0057] In RT-PCR, primers consisting of the nucleotide sequences described in SEQ ID NO: 1 and SEQ ID NO: 2 were used. After 40 cycles of PCR reaction, agarose gel electrophoresis was performed. A positive result was determined if a visible band was detected, and a negative result was determined if no visible band was detected. In addition, the incidence of gastric cancer in each patient after Helicobacter pylori eradication was followed up for 5 years.

[0058] Figure 1 is a graph showing the 5-year cumulative incidence rate of gastric cancer in the KK-LC-1 positive and negative groups. The results showed that the 5-year cumulative incidence rate of gastric cancer was 43% in the KK-LC-1 positive group. In contrast, the 5-year cumulative incidence rate of gastric cancer was 1% in the KK-LC-1 negative group.

[0059] These results indicate that if the expression of the KK-LC-1 gene is positive in gastric tissue samples from patients before Helicobacter pylori eradication, those patients are at risk of developing gastric cancer after eradication.

[0060] [Experimental Example 2] (Study using real-time RT-PCR) In Experimental Example 1, cancer cases were observed even in the KK-LC-1 negative group. Therefore, KK-LC-1 gene expression was detected using more sensitive real-time RT-PCR.

[0061] In 96 patients who underwent Helicobacter pylori eradication, gastric tissue samples collected before eradication were subjected to real-time RT-PCR to detect the expression of the KK-LC-1 gene.

[0062] In real-time RT-PCR, primers consisting of the nucleotide sequence described in SEQ ID NO: 1 and SEQ ID NO: 2 were used, along with a qPCR probe (named "MGB-D") consisting of the nucleotide sequence described in SEQ ID NO: 8. MGB-D was a qPCR probe modified with a fluorescent substance at its 5' end and a quenching substance and a minor groove binder (MGB) at its 3' end. MGB increases the Tm value.

[0063] The difference between the β-actin Ct value and the KK-LC-1 gene Ct value was calculated and defined as ΔCt. Based on the ΔCt value, participants were classified into KK-LC-1 high expression, KK-LC-1 medium expression, and KK-LC-1 low expression groups. Specifically, a ΔCt of -2 or higher was classified as the high expression group. A ΔCt of -5 or higher but less than -2 was classified as the medium expression group. A ΔCt of less than -5 was classified as the low expression group.

[0064] Figure 2 is a graph showing the 5-year cumulative incidence rate of gastric cancer in patients in each group. The results show that the 5-year cumulative incidence rate of gastric cancer was 40% in the high-KK-LC-1 expression group, and 5% in the medium-expression group. In contrast, the 5-year cumulative incidence rate of gastric cancer was 0% in the low-KK-LC-1 expression group. This result indicates that the detection sensitivity of KK-LC-1 has increased with highly sensitive real-time RT-PCR. Furthermore, it suggests that the expression level of the KK-LC-1 gene in gastric tissue samples from patients before Helicobacter pylori eradication correlates with the risk of developing gastric cancer after eradication. It also indicates that if the expression level of the KK-LC-1 gene in gastric tissue samples from patients before Helicobacter pylori eradication is low (ΔCt less than -5), the risk of developing gastric cancer after eradication can be considered low.

[0065] [Experimental Example 3] (QPCR probe evaluation 1) Plasmids containing genomic DNA and cDNA of the KK-LC-1 gene were amplified by real-time PCR using primers consisting of the nucleotide sequences described in Sequence ID No. 1 and Sequence ID No. 2, and further using the qPCR probes shown in Table 1 below. The concentration of the qPCR probe in the real-time PCR reaction mixture was 0.25 μM.

[0066] [Table 1]

[0067] Table 1 shows the presence or absence of MGB (Minor Groove Binder), the number of mutations, and the base length for each qPCR probe. All qPCR probes shown in Table 1 had base sequences containing the 195th and 196th bases of the KK-LC-1 gene cDNA base sequence (SEQ ID NO: 3). Therefore, all qPCR probes shown in Table 1 hybridized to the junction site between the first and second exons of the KK-LC-1 gene cDNA. In addition, the qPCR probes shown in Table 1 had 1 to 4 base mutations relative to the KK-LC-1 gene cDNA base sequence (SEQ ID NO: 3). In Table 1, the number of mutations refers to the number of mutations relative to SEQ ID NO: 3.

[0068] For comparison, a commercially available qPCR probe for the KK-LC-1 gene cDNA (name "Hs02386421_g1", Thermo Fisher Scientific) was also used. Hs02386421_g1 was a qPCR probe modified with a fluorescent substance at the 5' end and a quenching substance and MGB at the 3' end. The nucleotide sequence of Hs02386421_g1 is not publicly available from the manufacturer, but no mutations have been introduced into the nucleotide sequence of the KK-LC-1 gene cDNA.

[0069] Figure 3 is a graph showing the results of real-time PCR using a plasmid containing the KK-LC-1 gene cDNA as a template. The qPCR probe must react with the KK-LC-1 gene cDNA. In Figure 3, the vertical axis shows fluorescence intensity (relative value), and the horizontal axis shows the number of cycles. The graph also shows the amount of plasmid containing the KK-LC-1 gene cDNA used as a template.

[0070] The results showed that MGB-A and MGB-B did not react to the cDNA of the KK-LC-1 gene. In contrast, MGB-C and MGB-D showed responsiveness to the cDNA of the KK-LC-1 gene.

[0071] Figure 4 is a graph showing the results of real-time PCR using genomic DNA as a template. In Figure 4, the vertical axis represents fluorescence intensity (relative value), and the horizontal axis represents the number of cycles. The graph also shows the amount of genomic DNA used as the template. Genomic DNA from the human gastric cancer cell line H-111-TC was used as the genomic DNA. It is preferable that the qPCR probe does not react with genomic DNA.

[0072] As a result, it was confirmed that a commercially available qPCR probe for the KK-LC-1 gene cDNA (named "Hs02386421_g1") also reacts with genomic DNA. On the other hand, it was confirmed that MGB-C and MGB-D do not react with genomic DNA.

[0073] Figure 5 is a graph showing the results of real-time PCR using cDNA as a template. In Figure 5, the vertical axis represents fluorescence intensity (relative value), and the horizontal axis represents the number of cycles. The graph also shows the amount of plasmid containing the KK-LC-1 gene cDNA used as a template. The qPCR probe must react with the KK-LC-1 gene cDNA.

[0074] As a result, as shown in Figure 5 (left), Non-MGB-E and Non-MGB-F reacted to the cDNA of the KK-LC-1 gene with Ct values ​​equivalent to those of MGB-D. Furthermore, as shown in Figure 5 (right), Non-MGB-E and Non-MGB-F showed higher sensitivity compared to MGB-D.

[0075] Figure 6 is a graph showing the results of real-time PCR using genomic DNA as a template. In Figure 6, the vertical axis represents fluorescence intensity (relative value), and the horizontal axis represents the number of cycles. The graph also shows the amount of genomic DNA used as the template. Genomic DNA from the human gastric cancer cell line H-111-TC was used as the genomic DNA. It is preferable that the qPCR probe does not react with genomic DNA.

[0076] As a result, it was confirmed that when 10 ng of genomic DNA was used, MGB-D and Non-MGB-E also reacted with genomic DNA. On the other hand, Non-MGB-F showed low reactivity to genomic DNA.

[0077] [Experimental Example 4] (QPCR probe evaluation 2) Plasmids containing genomic DNA and cDNA of the KK-LC-1 gene were amplified by real-time PCR using primers consisting of the nucleotide sequence described in SEQ ID NO: 1, primers consisting of the nucleotide sequence described in SEQ ID NO: 2, the qPCR probe MGB-D shown in Table 1 above, and the single-stranded nucleic acid fragment shown in Table 2 below. The concentration of the qPCR probe in the real-time PCR reaction mixture was 0.25 μM. The concentration of the single-stranded nucleic acid fragment in the real-time PCR reaction mixture was 1.0 μM.

[0078] [Table 2]

[0079] Table 2 shows the base lengths for each single-stranded nucleic acid fragment. The single-stranded nucleic acid fragments shown in Table 2 suppressed the amplification of genomic DNA. All of the single-stranded nucleic acid fragments shown in Table 2 had base sequences containing the 322nd and 323rd bases in the base sequence (SEQ ID NO: 4) of the KK-LC-1 genomic DNA. Therefore, all of the single-stranded nucleic acid fragments shown in Table 2 hybridized to the intron-exon junction site in the KK-LC-1 genomic DNA.

[0080] Figure 7 is a graph showing the results of real-time PCR using genomic DNA as a template. In Figure 7, the vertical axis represents fluorescence intensity (relative value), and the horizontal axis represents the number of cycles. The graph also shows the amount of genomic DNA used as the template. Genomic DNA from the human gastric cancer cell line H-111-TC was used as the genomic DNA. It is preferable that the qPCR probe does not react with genomic DNA.

[0081] As a result, it was confirmed that MGB-D reacts to genomic DNA when 25 ng of genomic DNA is used. On the other hand, it was revealed that the reaction of MGB-D to genomic DNA is suppressed when one of Block1, Block2, or Block3 is present with MGB-D.

[0082] Figure 8 is a graph showing the results of real-time PCR using cDNA as a template. In Figure 8, the vertical axis represents fluorescence intensity (relative value), and the horizontal axis represents the number of cycles. The graph also shows the amount of plasmid containing the KK-LC-1 gene cDNA used as a template. The qPCR probe must react with the KK-LC-1 gene cDNA.

[0083] The results showed that the coexistence of Block1, Block2, or Block3 with MGB-D did not adversely affect the reactivity of MGB-D. Rather, as shown in Figure 8 (right), the coexistence of Block1, Block2, or Block3 with MGB-D increased the detection sensitivity of the KK-LC-1 gene cDNA.

[0084] [Experimental Example 5] (QPCR probe evaluation 3) Plasmids containing genomic DNA and cDNA of the KK-LC-1 gene were amplified by real-time PCR using primers consisting of the nucleotide sequence described in SEQ ID NO: 1, primers consisting of the nucleotide sequence described in SEQ ID NO: 2, the qPCR probes Non-MGB-E or Non-MGB-F shown in Table 1, and the single-stranded nucleic acid fragment Block 1 shown in Table 2. The concentration of the qPCR probe in the real-time PCR reaction mixture was 0.25 μM. The concentration of the single-stranded nucleic acid fragment in the real-time PCR reaction mixture was 1.0 μM. For comparison, a group using MGB-D as the qPCR probe was also prepared.

[0085] Figure 9 is a graph showing the results of real-time PCR using cDNA as a template. In Figure 9, the vertical axis represents fluorescence intensity (relative value), and the horizontal axis represents the number of cycles. The graph also shows the amount of plasmid containing the KK-LC-1 gene cDNA used as a template. The qPCR probe must react with the KK-LC-1 gene cDNA.

[0086] The results showed that the coexistence of Block1 with Non-MGB-E or Non-MGB-F did not adversely affect reactivity.

[0087] The above results indicate that the qPCR probes shown in Table 1, or combinations of the qPCR probes shown in Table 1 and the single-stranded nucleic acid fragments shown in Table 2, are suitable for detecting the expression of the KK-LC-1 gene.

[0088] [Experimental Example 6] (QPCR probe evaluation 4) Genomic DNA was amplified by real-time PCR using primers consisting of the nucleotide sequence described in SEQ ID NO: 1, primers consisting of the nucleotide sequence described in SEQ ID NO: 2, the qPCR probes MGB-D, Non-MGB-E, Non-MGB-F shown in Table 1, and Block1, a single-stranded nucleic acid fragment shown in Table 2.

[0089] For comparison, a commercially available qPCR probe for the KK-LC-1 gene cDNA (name "Hs02386421_g1", Thermo Fisher Scientific) was also used. Hs02386421_g1 was a qPCR probe modified with a fluorescent substance at the 5' end and a quenching substance and MGB at the 3' end. The nucleotide sequence of Hs02386421_g1 is not publicly available from the manufacturer, but no mutations have been introduced into the nucleotide sequence of the KK-LC-1 gene cDNA.

[0090] The concentration of the qPCR probe in the real-time PCR reaction mixture was 0.25 μM. The concentration of the single-stranded nucleic acid fragment in the real-time PCR reaction mixture was 1.0 μM.

[0091] Figures 10 and 11 are graphs showing the results of real-time PCR using genomic DNA as a template. In Figures 10 and 11, the vertical axis represents fluorescence intensity (relative value), and the horizontal axis represents the number of cycles. The graphs also show the amount of genomic DNA used as a template. Genomic DNA from the human gastric cancer cell line H-111-TC was used as the genomic DNA. It is preferable that the qPCR probe does not react with genomic DNA.

[0092] As a result, it was confirmed that Hs02386421_g1, MGB-D, Non-MGB-E, and Non-MGB-F also react with genomic DNA. On the other hand, it was found that the reaction to genomic DNA was suppressed when Block1 was present with these qPCR probes.

[0093] [Experimental Example 7] (Study using clinical specimens) Cancer tissue samples from breast cancer patients and cancer tissue samples from colorectal cancer patients were subjected to endpoint RT-PCR and real-time RT-PCR, respectively, to detect the expression of the KK-LC-1 gene.

[0094] In the endpoint RT-PCR, primers consisting of the nucleotide sequences described in SEQ ID NO: 1 and SEQ ID NO: 2 were used. After 40 cycles of PCR reaction, agarose gel electrophoresis was performed. A positive result was determined if a visible band was detected, and a negative result was determined if no visible band was detected.

[0095] Real-time RT-PCR was performed using primers consisting of the nucleotide sequences described in SEQ ID NO: 1 and SEQ ID NO: 2, and the qPCR probe MGB-D shown in Table 1 above. Real-time RT-PCR was also performed in or without Block 1, a single-stranded nucleic acid fragment shown in Table 2 above. The difference between the Ct value of β-actin and the Ct value of the KK-LC-1 gene was calculated and defined as ΔCt.

[0096] Table 3 below shows the results using cancer tissue samples from breast cancer patients, and Table 4 below shows the results using cancer tissue samples from colorectal cancer patients. As a result, with the exception of sample number 7 from a colorectal cancer patient, KK-LC-1 gene expression could be detected by real-time RT-PCR even when it was below the detection limit by endpoint RT-PCR. In sample number 7 from a colorectal cancer patient, KK-LC-1 gene expression was not detected by either endpoint RT-PCR or real-time RT-PCR.

[0097] In samples 8 and 9 from breast cancer patients and samples 3, 5, 6, and 8 from colorectal cancer patients, KK-LC-1 gene expression was detected in the absence of Block1, but not in the presence of Block1. This result was thought to be a false positive result due to the qPCR probe reacting to genomic DNA in the absence of Block1. In the presence of Block1, the false positive was suppressed, and accurate KK-LC-1 gene expression was detected.

[0098] [Table 3]

[0099] [Table 4] [Industrial applicability]

[0100] The present invention provides a technology for evaluating the risk of developing gastric cancer in patients after eradication of Helicobacter pylori.

Claims

1. A method for evaluating the risk of developing gastric cancer in patients after eradication of Helicobacter pylori, The process includes a step of detecting the expression of the KK-LC-1 gene in a gastric tissue sample derived from the patient before eradication of Helicobacter pylori, A method for demonstrating that positive expression of the KK-LC-1 gene indicates a risk of the patient developing gastric cancer after eradication of Helicobacter pylori.

2. The method according to claim 1, wherein the expression of the KK-LC-1 gene is detected by endpoint RT-PCR.

3. The method according to claim 2, wherein in the endpoint RT-PCR, a primer consisting of the nucleotide sequence described in SEQ ID NO: 1 and a primer consisting of the nucleotide sequence described in SEQ ID NO: 2 are used.

4. The method according to claim 1, wherein the expression of the KK-LC-1 gene is detected by real-time RT-PCR.

5. In the aforementioned real-time RT-PCR, a qPCR probe is used, and the qPCR probe is It has a nucleotide sequence that includes the 195th and 196th nucleotides in the nucleotide sequence described in Sequence ID No. 3, The base length is 15 to 50 bases. The method according to claim 4, wherein the sequence has one or two base mutations relative to the sequence described in Sequence ID No.

3.

6. The real-time RT-PCR described above is performed in the presence of a single-stranded nucleic acid fragment that suppresses the amplification of genomic DNA, and the single-stranded nucleic acid fragment is The method according to claim 4 or 5, comprising a sequence of 20 to 50 consecutive bases in the sequence of bases described in Sequence ID No. 4, including the 322nd and 323rd bases, or the 1096th and 1097th bases, or a sequence of bases complementary to said sequence.

7. The method according to claim 1, wherein the expression of the KK-LC-1 gene is detected at the protein level.

8. A kit for evaluating the risk of developing gastric cancer in patients after eradication of Helicobacter pylori, comprising a primer consisting of the nucleotide sequence described in SEQ ID NO: 1 and a primer consisting of the nucleotide sequence described in SEQ ID NO:

2.

9. The qPCR probe further comprises, It has a nucleotide sequence that includes the 195th and 196th nucleotides in the nucleotide sequence described in Sequence ID No. 3, The base length is 15 to 50 bases. The kit according to claim 8, having one or two base mutations in the base sequence described in Sequence ID No.

3.

10. It further comprises a single-stranded nucleic acid fragment that suppresses the amplification of genomic DNA, wherein the single-stranded nucleic acid fragment The kit according to claim 8 or 9, comprising a sequence of 20 to 50 consecutive bases from the sequence of bases described in Sequence ID No. 4, including the 322nd and 323rd bases, or the 1096th and 1097th bases, or a sequence of bases complementary to said sequence.

11. A kit for evaluating the risk of developing gastric cancer in patients after eradication of Helicobacter pylori, comprising a specific binding agent for the KK-LC-1 protein.

12. It has a nucleotide sequence that includes the 195th and 196th nucleotides in the nucleotide sequence described in Sequence ID No. 3, The base length is 15 to 50 bases. A polynucleotide having one or two base mutations from the base sequence described in Sequence ID No.

3.

13. A polynucleotide having a sequence of 20 to 50 consecutive bases from the sequence of bases described in Sequence ID No. 4, including the 322nd and 323rd bases, or the 1096th and 1097th bases, or a sequence of bases complementary to said sequence.

14. A method for detecting the expression of the KK-LC-1 gene in a biological sample, The above detection is performed by real-time RT-PCR, and in the real-time RT-PCR, a qPCR probe is used. The aforementioned real-time RT-PCR is performed in the presence of single-stranded nucleic acid fragments that suppress the amplification of genomic DNA. The single-stranded nucleic acid fragment is A method comprising a sequence of 20 to 50 consecutive bases from the sequence of bases described in Sequence ID No. 4, including the 322nd and 323rd bases, or the 1096th and 1097th bases, or a sequence of bases complementary to said sequence.

15. The aforementioned qPCR probe It has a nucleotide sequence that includes the 195th and 196th nucleotides in the nucleotide sequence described in Sequence ID No. 3, The base length is 15 to 50 bases. The method according to claim 14, wherein the sequence has one or two base mutations relative to the sequence described in Sequence ID No. 3.

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