JP-7856341-B2 - Method, polynucleotide, and kit for evaluating the risk of developing gastric cancer in patients after Helicobacter pylori eradication.

Inventors

• 福山 隆
• 坂口 佳奈子
• 小林 憲忠
• 山▲崎▼ 大賀
• 高橋 禎人
• 山▲崎▼ 等
• 二渡 信江
• 大塚 俊和
• 小泉 和三郎
• 市来 嘉伸

Assignees

• 学校法人北里研究所

Dates

Publication Date

20260511

Application Date

20230831

Priority Date

20220902

Claims (15)

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 Helicobacter pylori eradication, 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 Helicobacter pylori eradication, 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.

Description

This invention relates to a method, polynucleotide, and kit for evaluating the risk of developing gastric cancer in patients after eradication of Helicobacter pylori. This application claims priority under Japanese Patent Application No. 2022-140097, filed in Japan on September 2, 2022, the contents of which are incorporated herein by reference. Helicobacter pylori infection is known to be one of the causes of gastric cancer. Currently, if Helicobacter pylori infection is detected, eradication through oral medication is covered by insurance. It has been reported that the risk of developing gastric cancer is 1/3 in cases where Helicobacter pylori is eradicated compared to the group that does not undergo eradication. 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 to assess the risk of developing gastric cancer after Helicobacter pylori eradication. In other words, even if eradication is successful, the patient remains anxious about developing cancer, just as they were before eradication. Furthermore, because there is no cancer risk indicator after eradication, regular checkups are necessary for all cases of successful eradication, and this does not alleviate the burden on healthcare economics or medical resources. The risk indicators mentioned above are an unmet medical need that is strongly desired by both healthcare professionals and patients. Incidentally, cancer/testicular antigens are a general term for proteins that are not expressed anywhere other than cancer cells and testes. Kitakyushu lung cancer antigen-1 (KK-LC-1) is one type of cancer/testicular antigen. The inventors have previously shown that KK-LC-1 can be used as a marker expressed in various cancers (see, for example, Patent Document 1). Patent No. 6028253 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 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 is a graph showing the results of real-time PCR in Experiment Example 3.Figure 4 is a graph showing the results of real-time PCR in Experiment Example 3.Figure 5 is a graph showing the results of real-time PCR in Experimental Example 3.Figure 6 is a graph showing the results of real-time PCR in Experiment Example 3.Figure 7 is a graph showing the results of real-time PCR in Experimental Example 4.Figure 8 is a graph showing the results of real-time PCR in Experimental Example 4.Figure 9 is a graph showing the results of real-time PCR in Experimental Example 5.Figure 10 is a graph showing the results of real-time PCR in Experimental Example 6.Figure 11 is a graph showing the results of real-time PCR in Experimental Example 6. [Method for assessing the risk of developing gastric cancer in patients after Helicobacter pylori eradication] In one embodiment, the present invention provides 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 before 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. The inventors discovered that positive expression of the KK-LC-1 gene in gastric tissue samples from patients before Helicobacter pylori eradication indicates a risk of developing gastric cancer after eradication, and thus completed the present invention. The NCBI accession number for the human KK-LC-1 gene cDNA is NM_001017978.4. The NCBI accession number for the human KK-LC-1 gene genomic DNA is NC_000023.11. The NCBI accession number for the human KK-LC-1 protein is NP_001017978.1. As will be described later in the examples, the method of this embodiment can be used to evaluate the risk of developing gastric cancer in patients after eradication of Helicobacter pylori. Previously, there was no technology to evaluate the risk of developing gastric cancer after eradication of Helicobacter pylori. Gastric tissue samples collected at the time of Helicobacter pylori infection confirmation are preferably used as gastric tissue samples. A positive expression of the KK-LC-1 gene indicates that the patient is at risk of developing gastric cancer even after Helicobacter pylori eradication. In this case, measures such as increasing the frequency of gastric cancer screenings for the patient can be taken. A negative expression of the KK-LC-1 gene indicates a low risk of developing gastric cancer in patients who have undergone Helicobacter pylori eradication. In this case, the frequency of gastric cancer screenings for these patients can be reduced,