TWI753455B - Method for assessing risk of a subject suffering from gastric cancer or precancerous lesions, kit, analyzer, and biomarkerthereof - Google Patents

Abstract

A method for assessing the risk of a subject suffering from gastric cancer or precancerous lesions is provided, comprising the steps of: providing a sample obtained from the subject; measuring the methylation degree of a target gene in the sample, wherein the target gene is SPG20; and analyzing the methylation degree of a target gene, if the target gene is methylated, the subject is assessed as having a risk of gastric cancer or precancerous lesions. A kit, an analyzer and a biomarker for assessing the risk of a subject suffering from gastric cancer or precancerous lesions are also provided to achieve early detection of gastric cancer or precancerous lesions.

Description

Methods for assessing the risk of an individual having gastric cancer or precancerous lesions, kits thereof, analyzers thereof, and biomarkers thereof

The present disclosure relates to a method for assessing an individual's risk of developing cancer or precancerous lesions, a kit thereof, an analyzer and biomarkers thereof, and in particular, a method for assessing an individual's risk of developing gastric cancer or precancerous lesions, and a kit thereof groups, their analyzers and their biomarkers.

Gastric cancer is the leading cause of cancer death worldwide. Despite advances in the modalities of cancer treatment, the 5-year survival rate for gastric cancer patients remains below 15%, likely due to a lack of biomarkers for early detection.

Epigenetic modifications, including DNA methylation, play important regulatory roles in transcription and the development of embryos and disease. Due to the stability of methylation, methylation of cytosines of adjacent cytosine and guanine (CG) dinucleotides has been found in tissues and body fluids such as plasma and serum. These properties make DNA methylation an attractive target for non-invasive detection of cancer using cell free DNA (cfDNA).

DNA methylation can be detected in bodily fluids of a variety of human diseases, including cancer. However, there is no non-invasive and sensitive biomarker for early detection of gastric cancer, so the existing technology needs to be improved.

One embodiment of the present disclosure aims to provide a method for assessing the risk of an individual suffering from gastric cancer or precancerous lesions, a kit thereof, and an analyzer thereof, so as to provide non-invasive and sensitive biomarkers that can achieve early stage The effect of detecting gastric cancer or precancerous lesions.

An embodiment of the present disclosure provides a method for assessing the risk of an individual suffering from gastric cancer or precancerous lesions, comprising the following steps: providing a sample obtained from the individual; measuring the methylation level of a target gene in the sample, wherein the target gene is SPG 20; and analyzing the degree of methylation of the target gene, if the target gene has a methylation status, the individual is assessed as having a risk of developing gastric cancer or precancerous lesions.

Another embodiment of the present disclosure provides a method for assessing the risk of an individual suffering from gastric cancer or precancerous lesions, comprising the steps of: providing a sample obtained from the individual; measuring the methylation level of a target gene in the sample, wherein the target gene SPG 20; and compare the methylation level of the same target gene in the control group and the sample, when the methylation level of the target gene in the sample is higher than the methylation level of the target gene in the control group, the individual is evaluated as having gastric cancer or risk of precancerous lesions.

In some embodiments, the control group is a sample taken from a normal person, comprising blood, urine, tears, ascites, feces, sputum, gastric juice, ex vivo gastric tissue, or a combination thereof.

In some embodiments, the detection of the methylation status of the target gene is methylation-specific polymerase chain reaction (methylation-specific PCR, MSP), quantitative methylation-specific polymerase chain reaction (quantitative methylation-specific PCR, QMSP), bisulfite sequencing (BS), microarray (microarray), mass spectrometer (MS), or pyrosequencing.

In some embodiments, the target gene SPG 20 has the nucleotide sequence shown in SEQ ID No:1.

In some embodiments, the SPG 20 methylation status of the target gene is detected by the primer pair nucleotide sequences shown in SEQ ID NOs: 2 and 3, or 5 and 6.

In some embodiments, the target gene SPG 20 comprises a plurality of CpG sequences located in the segment from +10 to +91 region of the transcription start site of SPG 20.

In some embodiments, the precancerous lesion comprises intestinal metaplasia (IM).

In some embodiments, the sample comprises blood, urine, tears, ascites, feces, sputum, gastric juice, ex vivo gastric tissue, or a combination thereof.

Another embodiment of the present disclosure provides a kit for assessing the risk of an individual suffering from gastric cancer or precancerous lesions, comprising: a target gene methylation primer mixture, comprising a first forward primer and a first reverse primer, The sequences of the first forward primer and the first reverse primer are selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 2 90% to about 99% identical sequences, about 90% to about 99% identical sequences to SEQ ID NO:3, about 90% to about 99% identical sequences to SEQ ID NO:5, and SEQ ID NO:6 sequences with about 90% to about 99% similarity, complementary strands of SEQ ID NO:2, complementary strands of SEQ ID NO:3, complementary strands of SEQ ID NO:5, and SEQ ID NO: The group consisting of 6 complementary strands; the target gene has no methylation mixture, including the second forward primer and the second reverse primer, and the sequences of the second forward primer and the second reverse primer are selected from SEQ ID NO: 7, SEQ ID NO: 8, a sequence having about 90% to about 99% similarity to SEQ ID NO: 7, a sequence having about 90% to about 99% similarity to SEQ ID NO: 8, The group consisting of the complementary strand of SEQ ID NO:7 and the complementary strand of SEQ ID NO:8; and a polymerase chain amplification reaction mixture comprising polymerase, deoxy-ribonucleoside triphosphate (deoxy-ribonucleoside triphosphate, dNTP) and magnesium salts.

In some embodiments, the kit further comprises probes selected from the group consisting of SEQ ID NO: 4, a sequence having about 90% to about 99% similarity to SEQ ID NO: 4, and SEQ ID NO: 4 A group consisting of complementary stocks of NO:4.

In some embodiments, the set further comprises an internal control gene detection mixture, comprising a third forward primer and a third reverse primer, and the internal control gene is selected from at least one or more of the following genes: A group consisting of GAPDH, β-actin, Col2A, and β-Globin.

In some embodiments, the internal control gene is GAPDH, and the sequences of the third forward primer and the third reverse primer are selected from the group consisting of SEQ ID NO:9, SEQ ID NO:10, and SEQ ID NO:9 A sequence of 90% to about 99% similarity, a sequence of about 90% to about 99% similarity to SEQ ID NO: 10, the complementary strand of SEQ ID NO: 9, and the complementary strand of SEQ ID NO: 10. 's group.

Another embodiment of the present disclosure provides an analyzer for evaluating the risk of an individual suffering from gastric cancer or precancerous lesions, comprising: a detection device for detecting the methylation level of a target gene in a sample obtained from the individual, wherein the target gene It is SPG 20; the computing device calculates the methylation degree of the target gene to obtain the calculation result; and the determination device analyzes the calculation result, if the target gene has a methylation state, the individual is evaluated as suffering from gastric cancer or precancerous lesions risks of.

Another embodiment of the present disclosure provides an analyzer for assessing the risk of an individual suffering from gastric cancer or precancerous lesions, comprising: a detection device for detecting the methylation level of a target gene in a sample obtained from the individual, wherein the target gene It is SPG 20; a computing device calculates and compares the same target gene methylation degree of the control group and the sample, and obtains the calculation result; and a judgment device, analyzes the calculation result, when the methylation degree of the target gene of the sample is higher than the target gene of the control group The degree of methylation of the gene, the individual is assessed as being at risk of developing gastric cancer or precancerous lesions.

In some embodiments, the target gene SPG 20 has the nucleotide sequence shown in SEQ ID No:1.

In some embodiments, the methylation status of the target gene SPG 20 is detected by the nucleotide sequences of primer pairs as shown in SEQ ID NOs: 2 and 3, or 5 and 6.

In some embodiments, the target gene SPG 20 comprises a plurality of CpG sequences located in the segment from +10 to +91 region of the transcription start site of SPG 20.

Another embodiment of the present disclosure provides a biomarker for assessing an individual's risk of developing gastric cancer or precancerous lesions, comprising methylation of at least one CpG sequence on the SPG 20 gene, the CpG sequence being located at the transcription start site of SPG 20 Point the section from +10 to +91 area.

In some embodiments, the biomarker further comprises methylation of a plurality of CpG sequences located in the region +10 to +91 of the transcription start site of SPG 20.

In order to make the description of the present disclosure more detailed and complete, the following provides an illustrative description of the implementation aspects and specific embodiments of the present disclosure, but this is not the only form of implementing or using the specific embodiments of the present disclosure. The embodiments disclosed below can be combined or substituted with each other under beneficial circumstances, and other embodiments can also be added to one embodiment without further description or explanation. In the following description, numerous specific details are set forth in detail to enable the reader to fully understand the following embodiments. However, embodiments of the present disclosure may also be practiced without these specific details.

As used herein, unless the context specifically defines the article, "a" and "the" can refer to a single one or a plurality. It will be further understood that the terms "comprising", "including", "having" and similar words used herein designate the recited features, regions, integers, steps, operations, elements and/or components, but do not exclude one or more of its other features, regions, integers, steps, operations, elements, components, and/or groups thereof, described or additional thereto.

In the following evaluation process, there may be one or more additional operational steps among/between the described steps, and the order of operational steps may be changed.

As used herein, the term "precancerous lesions" is used herein to refer to the abnormal proliferation of cells in certain organs and tissues of the human body. These abnormal proliferations are intermediate stages in the transition of cells from benign to malignant lesions, because there may be deteriorations that develop into Predisposition to cancer.

As used herein, "gastritis" refers to an inflammatory reaction within the stomach lining, which may be short and intense, or a long-lasting process. Common causes of gastritis include Helicobacter pylori infection and use of non-steroidal anti-inflammatory drugs (NSAIDs); relatively rare causes of gastritis include alcohol consumption, smoking, cocaine use, autoimmune diseases, radiation therapy, cloning schizophrenia and other serious diseases.

In this article, "intestinal metaplasia (IM)", also known as gastric intestinal metaplasia, refers to the fact that the normal gastric mucosa is damaged due to chronic inflammation, and part of the result is transformed to resemble the "intestinal cell shape". After a long time, there is a chance of developing stomach cancer.

In this paper, "detection" and "screening" are not deterministic diagnostic methods. According to the medical knowledge in the prior art and the information obtained by detecting the methylation status of target genes in various types of samples, the information itself , does not directly lead to the diagnosis or health status, that is, whether there is a diagnosis of cancer, but only belongs to "processing or testing of tissues, body fluids or excreta that have been separated from the human or animal body to obtain as an intermediate result. A method of information, or a method of processing that information", is an "invention that is not a method of diagnosis". According to those with ordinary knowledge in the art (eg, doctors, medical researchers), it is well known that pathological section and other methods must be performed to diagnose cancer.

Herein, a "site" corresponds to a single site, which can be a single base position or a group of related base positions, such as a CpG site.

In some embodiments, there is provided a method for assessing the risk of developing gastric cancer or precancerous lesions in an individual, comprising the steps of: providing a sample obtained from the individual; measuring the methylation level of a target gene in the sample, wherein the target gene is SPG 20; and analyzing the degree of methylation of the target gene, if the target gene has a methylation state, the individual is assessed as having a risk of developing gastric cancer or precancerous lesions.

Another embodiment of the present disclosure provides a method for assessing the risk of an individual suffering from gastric cancer or precancerous lesions, comprising the steps of: providing a sample obtained from the individual; measuring the methylation level of a target gene in the sample, wherein the target gene SPG 20; and compare the methylation level of the same target gene in the control group and the sample, when the methylation level of the target gene in the sample is higher than the methylation level of the target gene in the control group, the individual is evaluated as having gastric cancer or risk of precancerous lesions.

In one embodiment, the target gene SPG 20 is methylated when gastric cancer or precancerous lesions such as stomach develop intestinal metaplasia.

In one embodiment, the target gene SPG 20 comprises a plurality of CpG sequences located in the segment from +10 to +91 region of the transcription start site of SPG 20. In some embodiments, 6 CpG sequences are included.

In one embodiment, when the average methylation of 6 CpG sequences is calculated in the isolated tissue, the methylation degree from low to high is gastritis (similar to normal tissue around gastric cancer), mild intestinal metaplasia, intestinal Epithelial metaplasia, and gastric cancer. In some embodiments, the degree of methylation of SPG 20 in Asian gastric cancer tissue is higher than in non-Asian gastric cancer tissue.

In one embodiment, the nucleotide sequences shown in SEQ ID NOs: 5 and 6 are detected by primer pairs in the ex vivo tissue, wherein the cfDNA in gastric cancer cell lines or blood with gastric cancer is methylated , cfDNA in normal blood and cfDNA in blood with gastritis were not methylated. In some embodiments, the sample comprises blood, urine, tears, ascites, feces, sputum, gastric juice, ex vivo gastric tissue, or a combination thereof.

In some embodiments, a kit for assessing the risk of an individual suffering from gastric cancer or precancerous lesions is provided, comprising: a mixture of target gene methylation primers, comprising a first forward primer and a first reverse primer, a first The sequences of the forward primer and the first reverse primer include, but are not limited to, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 2 with about 90% Sequences to about 99% similarity, sequences about 90% to about 99% similar to SEQ ID NO: 3, sequences about 90% to about 99% similar to SEQ ID NO: 5, and SEQ ID NO: 5 NO:6 sequences having about 90% to about 99% similarity, complementary strands of SEQ ID NO:2, complementary strands of SEQ ID NO:3, complementary strands of SEQ ID NO:5, or complementary strands of SEQ ID NO:6 Complementary stock; the target gene has no methylation mixture, including the second forward primer and the second reverse primer, the sequences of the second forward primer and the second reverse primer include, but are not limited to SEQ ID NO: 7, SEQ ID NO: 7, SEQ ID NO: 7, SEQ ID NO: 7 ID NO: 8, a sequence about 90% to about 99% similar to SEQ ID NO: 7, a sequence about 90% to about 99% similar to SEQ ID NO: 8, the complement of SEQ ID NO: 7 strand, or the complementary strand of SEQ ID NO: 8; and a polymerase chain amplification reaction mixture, the main components of which at least comprise polymerase, deoxyribonucleoside triphosphate (dNTP) and magnesium salt.

In one embodiment, the kit further comprises probes including, but not limited to, SEQ ID NO: 4, a sequence with about 90% to about 99% similarity to SEQ ID NO: 4, or SEQ ID Complementary shares of NO:4.

In one embodiment, the probe has a fluorescent label selected from the group consisting of, but not limited to, FAM, HEX, TET, TAMRA, Cy3, Cy5, Cy5.5, VIC, Red610, Yellow 555, Texas Fluorescence of Red, Yakima Yellow, BHQ-1, BHQ-2 or BHQ-3.

In one embodiment, the polymerase chain amplification reaction mixture further comprises a fluorescent substance capable of recognizing the products of the chain amplification reaction or a substance selected from the group consisting of substances that can be double-stranded with DNA to generate fluorescence and then be detected. This substance includes, but is not limited to, SYBR series substances such as SYBER Green, Syber Gold, and the like.

In one embodiment, the set further comprises an internal control gene detection mixture, comprising the third forward primer and the third reverse primer, and the internal control gene includes, but is not limited to, GAPDH, β-actin, Col2A, or β-Globin. In one embodiment, the internal control gene is GAPDH, and the sequences of the third forward primer and the third reverse primer include, but are not limited to, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 9. A sequence of 90% to about 99% similarity, a sequence of about 90% to about 99% similarity to SEQ ID NO:10, the complementary strand of SEQ ID NO:9, or the complementary strand of SEQ ID NO:10.

In one embodiment, the sequences set forth in SEQ ID NOs: 2, 3, 4, 5, 6, 7, 8, 9, and 10 may also tolerate some degree of variation. That is, for example, sequences having a similarity of about 90% to about 99% with SEQ ID NOs: 2, 3, 4, 5, 6, 7, 8, 9, and 10, respectively, are used in this embodiment. the same effect. For example, primer pairs can be selected to include the degenerate sequences of SEQ ID NOs: 2, 3, 4, 5, 6, 7, 8, 9, and 10. As used herein, "degenerate sequences" refer to oligonucleotide sequences disclosed herein in which some nucleotides are replaced by other nucleotides. In other words, the degenerate sequence of SEQ ID NO: 2 means that the oligonucleotide of SEQ ID NO: 2 can be allowed to have a degree of variation of about 1% to about 10% under the condition that the length of the sequence of SEQ ID NO: 2 is unchanged, and other sequences can be used accordingly. analogy. In other embodiments, the selection of primer pairs may also comprise derived sequences of SEQ ID NOs: 2, 3, 4, 5, 6, 7, 8, 9, and 10. As used herein, "derived sequences" refer to oligonucleotide sequences disclosed herein that can be modified at the 3' or 5' ends and still retain part or all of the sequence. In other words, the derived sequence of SEQ ID NO: 2 means that under the condition that the length of the sequence of SEQ ID NO: 2 can be increased or decreased, its oligonucleotide can be allowed to have a degree of variation of about 1% to about 10%, and other sequences can be And so on.

In one embodiment, the bisulfite-converted plasma-free DNA can be captured first using a solution-phase or solid-phase hybridization-based process, followed by MPS. Large-scale sequencing can be performed using, for example, the sequencing-by-synthesis platform from Illumina®, the sequencing-by-joint platform for ^SOLiD® from Life Technologies®, the ^{Ion Torrent®} or ^{Ion Proton® semiconductor} sequencing systems from Life Technologies® , or a nanopore-based single-molecule sequencing system. Nanopore-based sequencing includes nanopores constructed using, for example, lipid bilayers and protein nanopores, and solid state nanopores (eg, graphene-based nanopores). Because the single-molecule sequencing platform chosen will allow direct discrimination of the methylation status of DNA molecules without bisulfite conversion.

In one embodiment, methylation analysis is performed on bisulfite-converted genomic DNA according to the ^Infinium® Methylation Analysis Protocol. Hybridization bead wafers were scanned on an Illumina ^® instrument, and DNA methylation was analyzed by methylation module software to normalize to an internal control and remove background. The methylation index of individual CpG sites is represented by the beta value, which is calculated using the ratio of fluorescence intensities between methylated and unmethylated alleles.

In some embodiments, there is provided a biomarker for assessing an individual's risk of developing gastric cancer or precancerous lesions, comprising methylation of at least one CpG sequence on the SPG 20 gene, the CpG sequence located at the transcription start site of SPG 20+ Section 10 to +91 area. Specifically, the CpG sequence includes one of +10, +15, +42, +45, +80, +90 of the transcription start site located at SPG 20.

In one embodiment, the biomarker comprises methylation of a plurality of CpG sequences on the SPG 20 gene, the CpG sequences are located in the segment +10 to +91 region of the transcription start site of SPG 20. Specifically, the CpG sequence includes two or more of the transcription initiation sites +10, +15, +42, +45, +80, and +90 located in SPG 20. In some embodiments, the biomarkers include methylation at the following sites for identification: transcription start sites +10 and +15, sites +10 and +42, sites +10 and +45, sites +10 and +45 at SPG 20 Any combination of positions +10 and +80, positions +10 and +90, positions +10, +15 and +42, positions +10, +15, +42 and +45, etc.

Although a series of operations or steps are used below to describe the methods disclosed herein, the order in which these operations or steps are shown should not be construed as a limitation of the present disclosure. For example, certain operations or steps may be performed in a different order and/or concurrently with other steps. Furthermore, not all illustrated operations, steps and/or features must be performed in order to implement embodiments of the present disclosure. Furthermore, each operation or step described herein may contain several sub-steps or actions.

Example 1

To further clarify the role of DNA methylation in the regulation of SPG 20 expression, evaluation was performed in normal gastric epithelial cell (GES) and a series of gastric cancer cell lines. A range of gastric cancer cell lines including AGS (ATCC ^® CRL-1739 ^™ ), KATO III (ATCC ^® HTB-103 ^™ ), MKN28, MKN45, SNU1 (ATCC ^® CRL-5971 ^™ ), SNU16 (ATCC ^® CRL-5974 ^™ ) All were purchased from ATCC. All cell lines were cultured in Roswell Park Memorial Institute medium (RPMI 1640) containing 10% Fetal Bovine Sera (FBS) and 1% Penicillin-Streptomycin and incubated at 37°C C, 5% _CO2 incubator. One batch of cells was collected on day 0 and RNA was extracted from all the above cell lines. Another batch of cells was administered with methyltransferase (DNA methyltransferase, DNMT) inhibitor 5aza-DC (5-aza-2'-deoxycytidine) 0.5μM to all gastric cancer cell lines, collected and extracted RNA on the third day to extract all the above RNA of the cell line, using glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as an internal control gene (primer pairs such as SEQ ID NOs: 9-10), to analyze the expression of the SPG 20 gene.

As shown in Figure 1, a significant decrease in RNA expression of SPG 20 was observed in all gastric cancer cell lines except normal gastric epithelial cells (GES). As shown in Figure 2, the RNA of SPG 20 was strongly re-expressed in most gastric cancer cell lines when the methyltransferase inhibitor 5aza-DC was administered.

Example 2

In order to further clarify the site of SPG 20 methylation, all gastric cancer cell lines and GES cell lines administered with the methyltransferase inhibitor 5aza-DC in Example 1 were extracted with bisulfite and transformed with bisulfite ( bisulphite conversion), followed by pyrosequencing of SEQ ID NOs: 2 and 3 and probe SEQ ID NO: 4 with primers, and the clipped sequence is shown in SEQ ID NO: 1.

As shown in Figure 3, the DNA of SPG 20 in these gastric cancer cell lines and GES cell lines administered with the methyltransferase inhibitor 5aza-DC was converted with bisulfite and pyrosequenced. The sequence SEQ ID NO: 1 includes 6 CpG sequences with significant methylation, which are located at +10, +15, +42, +45, +80, and +90 of the transcription initiation site of SPG 20, respectively. Therefore, SPG 20 RNA was strongly re-expressed in most gastric cancer cell lines when the methyltransferase inhibitor 5aza-DC was administered, which was associated with DNA demethylation in the SPG 20 promoter region.

Example 3

In order to further test whether it is consistent with the results of gastric cancer cell lines in clinical practice, isolated patient samples were obtained, including gastric samples from 34 patients with gastritis, normal tissue samples around tumors from 40 patients with gastric cancer, and 33 gastric intestinal epithelial samples. Raw (IM) samples and gastric samples from 53 gastric cancer patients. The above samples were subjected to the same bisulfite conversion and pyrosequencing method as in Example 2 to detect the methylation level of the SPG 20 gene.

As shown in Figure 4, after bisulfite pyrophosphate sequencing, it was shown that the degree of methylation of SPG 20 in gastric cancer specimens was higher than that in intestinal metaplasia tissue, normal tissue around the tumor, and gastritis tissue. It is worth noting that the degree of SPG 20 methylation in intestinal metaplasia tissue is also higher than that in the surrounding normal tissue and gastritis tissue.

Example 4

The analysis was performed from the two major databases, GSE103186 and TCGA, and the methylation microarray data were taken. The methylation level (β value) of each probe was calculated by the following formula:

As shown in FIG. 5 , the methylation levels of SPG 20 are in descending order of gastric cancer tissue, intestinal metaplasia tissue, mild intestinal metaplasia tissue, and normal gastric tissue. In gastric cancer tissues, the methylation level of Asians was higher than that of non-Asian.

Example 5

In order to further examine the feasibility of SPG 20 methylation as a non-invasive methylation biomarker, cell-free DNA from a series of gastric cancer cell lines, GES, and gastric cancer patient plasma (groups T1 and T2 in Example 1) were taken. , T6, T7, T9), cell-free DNA in the plasma of gastritis patients (groups are GS12, GS49), methylated positive control group (IVD, in vitro methylated DNA), plasma of non-cancer patients (NB, normal blood) as a negative control group, and water (H ₂ O) as a control group. The above groups were subjected to methylation-specific PCR (MSP), and the primer pair SEQ ID NOs: 5 and 6 were used to identify the methylated SPG 20 gene, and the primer pair SEQ ID NOs: 7 and 8 were used to identify the unmethylated gene. the SPG 20 gene.

As shown in Figure 6, in agreement with our bisulfite pyrosequencing results, SPG 20 methylation was detected in all gastric cancer cell lines by MSP detection. As shown in Fig. 7, SPG 20 methylation was also detected in cell-free DNA in the plasma of most gastric cancer patients, but no SPG 20 methylation was detected in cell-free DNA in the plasma of gastritis patients.

In addition, as shown in Table 1 below, in the detection of cell-free DNA in plasma, the sensitivity and specificity of detecting gastric cancer using SPG 20 methylation were 88.6% and 75%. It is worth mentioning that the sensitivity of cancer or intestinal metaplasia detection can still be as high as 87.5%. Table 1, type* Gastric cancer only (n = 53) Gastric cancer or IM (n = 53+3) Sensitivity 88.6% (47/53) 87.5% (49/56) specificity 75.0% (15/20) 75.0% (15/20) Positive Predictive Value (PPV) 90.3% 90.7% Negative Predictive Value (NPV) 71.4% 68.1% *Comparison with cell-free DNA in plasma from 20 non-cancer patients.

Example 6

Please refer to FIG. 8, which depicts a schematic diagram of an analyzer for assessing an individual's risk of developing peritoneal sclerosis. The analyzer 100 includes a detection device 110 , an operation device 120 and a determination device 130 .

(1) Calculated by methylation-specific PCR (MSP)

The detection device 110 detects the methylation level of the target gene in the sample obtained from the individual, wherein the target gene is SPG 20 . The detection method is as shown in Example 5, and the methylation degree of the target gene is detected by methylation-specific PCR.

The computing device 120 performs computing on the methylation degree of the target gene to obtain a computing result. The operation method is to compare the difference with the background value.

The determination device 130 analyzes the calculation result, and if the target gene has a methylation state, the individual is estimated to be at risk of developing gastric cancer or precancerous lesions.

(2) bisulfite conversion and pyrosequencing, microarray operation

The detection device 110 detects the methylation level of the target gene in the sample obtained from the individual, wherein the target gene is SPG 20 . The detection method is as described in Example 2, DNA is first transformed with bisulfite, and then pyrosequencing is performed; or DNA is first transformed with bisulfite, and then the microarray as described in Example 4 is used. detect.

The computing device 120 computes and compares the methylation level of the target gene in the control group and the sample to obtain a computing result. Such as calculating the percentage of target gene methylation degree, or β value. The control group is a sample taken from normal people, including blood, urine, tears, ascites, feces, sputum, gastric juice, isolated gastric tissue or a combination thereof.

The determination device 130 analyzes the calculation result, and when the methylation degree of the target gene in the sample is higher than the methylation degree of the target gene in the control group, the individual is estimated to be at risk of developing gastric cancer or precancerous lesions.

Although the present disclosure has been disclosed as above in embodiments, it is not intended to limit the present disclosure. Anyone skilled in the art can make various changes and modifications without departing from the spirit and scope of the present disclosure. Therefore, the present disclosure protects The scope shall be determined by the scope of the appended patent application.

100: Analyzer 110: Detection device 120: Computing device 130: Judgment Device

In order to make the above and other objects, features, advantages and embodiments of the present disclosure more clearly understood, the accompanying drawings are described as follows: Figure 1 illustrates the performance of SPG 20 according to an embodiment of the present disclosure in different cell lines Bar graph of amount; *** indicates p > 0.005 compared to GES. Figure 2 is a bar graph showing the expression of SPG 20 according to an embodiment of the present disclosure in different cell lines treated with DMSO or 5aza-DC; * means p > 0.05, *** means p > 0.005, ns means p > 0.05 No significant difference. Figure 3 is a line graph of the methylation ratios of 6 CpG sites in different cell lines according to an embodiment of the disclosure. Figure 4 is a scatter diagram of the methylation ratio of SPG 20 in different states of gastric tissue according to an embodiment of the disclosure; * means p>0.05, *** means p>0.005, ns means no significant difference. Fig. 5 is a box plot of the methylation ratio of SPG 20 in different states of gastric tissue according to an embodiment of the disclosure; * means p>0.05, *** means p>0.005. Fig. 6 is an electropherogram showing the presence or absence of methylation of SPG 20 in different cell lines or blood according to an embodiment of the disclosure. FIG. 7 is an electropherogram showing the presence or absence of methylation in gastric tissue or blood in different states of SPG 20 according to an embodiment of the disclosure. FIG. 8 is a schematic diagram of an analyzer for assessing an individual's risk of developing gastric cancer or precancerous lesions, according to an embodiment of the present disclosure.

Domestic storage information (please note in the order of storage institution, date and number) none Foreign deposit information (please note in the order of deposit country, institution, date and number) none

Claims (17)

A method for assessing the risk of an individual suffering from gastric cancer or precancerous lesions, comprising the steps of: providing a sample obtained from the individual, the sample being blood; measuring the methylation level of a target gene in the sample, wherein the The target gene is SPG 20, wherein the target gene SPG 20 comprises a plurality of CpG sequences, and these CpG sequences are located in the segment of the transcription initiation site +10 to +91 region of SPG 20; and analyze the methylation of the target gene If the target gene has methylation status, the individual is assessed to be at risk of developing gastric cancer or precancerous lesions. A method for assessing the risk of an individual suffering from gastric cancer or precancerous lesions, comprising the steps of: providing a sample obtained from the individual, the sample being blood; measuring the methylation level of a target gene in the sample, wherein the The target gene is SPG 20, wherein the target gene SPG 20 comprises a plurality of CpG sequences, and the CpG sequences are located in the segment of the transcription initiation site +10 to +91 region of SPG 20; and compare a control group with the sample For the same methylation degree of the target gene, when the methylation degree of the target gene in the sample is higher than the methylation degree of the target gene in the control group, the individual is evaluated as having a risk of developing gastric cancer or precancerous lesions. The method of claim 2, wherein the control group is a sample taken from a normal person, and the sample is blood. The method according to claim 1 or 2, wherein the detection of the methylation status of the target gene is methylation-specific polymerase chain reaction (methylation-specific PCR, MSP), quantitative methylation-specific polymerase chain reaction (quantitative methylation-specific PCR, QMSP), bisulfite sequencing (BS), microarray (microarray), mass spectrometer (MS), or pyrosequencing (pyrosequencing). The method according to claim 1 or 2, wherein the target gene SPG 20 has the nucleotide sequence shown in SEQ ID No: 1. The method of claim 1 or 2, wherein the SPG 20 methylation status of the target gene is detected by a primer pair of nucleotide sequences shown in SEQ ID NOs: 2 and 3, or 5 and 6. The method of claim 1 or 2, wherein the precancerous lesion comprises intestinal metaplasia (IM). A method used to evaluate an individual for gastric cancer or precancerous lesions The risk set, comprising: a target gene methylation primer mixture, comprising a first forward primer and a first reverse primer, the sequence of the first forward primer and the first reverse primer are selected Nucleotide sequences from the group consisting of SEQ ID NO: 2 and SEQ ID NO: 3, and SEQ ID NO: 5 and SEQ ID NO: 6, their complementary nucleotide sequences, their degenerate sequences or its derived sequence, wherein the degenerate sequence refers to more than 90% homology to the nucleotide sequences shown in SEQ ID NOs: 2, 3, 5 and 6, and the derived sequence refers to the 3' end or the 5' end Modified to have a nucleotide sequence with more than 90% homology with SEQ ID NOs: 2, 3, 5 and 6; a target gene without methylation mixture, comprising a second forward primer and a first Two reverse primers, the sequences of the second forward primer and the second reverse primer are selected from the nucleotide sequences of the group consisting of SEQ ID NO: 7 and SEQ ID NO: 8, their complements The nucleotide sequence, its degenerate sequence or its derivative sequence, wherein the degenerate sequence refers to more than 90% homology to the nucleotide sequences shown in SEQ ID NOs: 7 and 8, and the derivative sequence refers to the nucleotide sequence shown in 3 The 'end or 5' end is modified so that it has a nucleotide sequence with more than 90% homology with SEQ ID NOs: 7 and 8; and a polymerase chain amplification reaction mixture, comprising polymerase, deoxyribose nucleus glycoside triphosphate (deoxy-ribonucleoside triphosphate, dNTP) and magnesium salt. The kit of claim 8, further comprising a probe selected from the core group consisting of SEQ ID NO: 4 A nucleotide sequence, its complementary nucleotide sequence, its degenerate sequence or its derivative sequence, wherein the degenerate sequence refers to more than 90% homology to the nucleotide sequence shown in SEQ ID NO: 4, the derivative sequence Refers to a nucleotide sequence modified at the 3' end or 5' end to have more than 90% homology with SEQ ID NO: 4. The kit according to claim 8, further comprising an internal control gene detection mixture comprising a third forward primer and a third reverse primer, the internal control gene is selected from at least one or one of the following The above genes: GAPDH, β-actin, Col2A, and β-Globin group. The kit of claim 10, wherein the internal control gene is GAPDH, and the sequences of the third forward primer and the third reverse primer are selected from SEQ ID NO: 9 and SEQ ID NO: 10 The nucleotide sequence of the formed group, its complementary nucleotide sequence, its degenerate sequence or its derivative sequence, wherein the degenerate sequence refers to more than 90% homologous to those shown in SEQ ID NOs: 9 and 10 The derived sequence refers to a nucleotide sequence modified at the 3' end or 5' end so that it has more than 90% homology with SEQ ID NOs: 9 and 10. An analyzer for assessing the risk of an individual suffering from gastric cancer or precancerous lesions, comprising: a detection device for detecting the methylation level of a target gene in a sample obtained from the individual, wherein the target gene is SPG 20 , wherein the target gene SPG 20 comprises a plurality of CpG sequences, and these CpG sequences are located in the segment of the transcription initiation site +10 to +91 region of the SPG 20, wherein the sample is blood; a computing device, the target gene The degree of methylation is calculated to obtain a calculation result; and a determination device is used to analyze the calculation result. If the target gene has a methylation state, the individual is estimated to be at risk of developing gastric cancer or precancerous lesions. An analyzer for assessing the risk of an individual suffering from gastric cancer or precancerous lesions, comprising: a detection device for detecting the methylation level of a target gene in a sample obtained from the individual, wherein the target gene is SPG 20 , wherein the target gene SPG 20 includes a plurality of CpG sequences, and these CpG sequences are located in the segment of the transcription initiation site +10 to +91 region of the SPG 20, wherein the sample is blood; a computing device, computing and comparing A control group has the same methylation degree of the target gene as that of the sample to obtain an operation result; and a determination device analyzes the operation result, when the methylation degree of the target gene of the sample is higher than that of the target gene of the control group The degree of cytoplasmization, the individual is assessed to be at risk of developing gastric cancer or precancerous lesions. The analyzer of claim 12 or 13, wherein the target gene SPG 20 has the nucleotide sequence shown in SEQ ID No:1. According to the analyzer of claim 12 or 13, the methylation status of the target gene SPG 20 is detected by the primer pair nucleotide sequences shown in SEQ ID NOs: 2 and 3, or 5 and 6. A biomarker for assessing an individual's risk of developing gastric cancer or precancerous lesions, comprising methylation of at least one CpG sequence on the SPG 20 gene located in the region +10 to +91 of the transcription start site of SPG 20 section. The biomarker of claim 16, further comprising methylation of a plurality of CpG sequences located in the region from +10 to +91 of the transcription initiation site of SPG 20.

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