WO2024001602A1 - Composition for detecting gastric cancer, kit, and use thereof - Google Patents

Abstract

Provided is a composition for detecting gastric cancer, comprising: a detection reagent for detecting the methylation level in the following region: a region in OTX1 gene set forth in SEQ ID NO: 1; a region in ZNF671 gene set forth in SEQ ID NO: 2; and a region in ELMO1 gene set forth in SEQ ID NO: 3; or a detection reagent for detecting the methylation level in the following region: a region in KCNA3 gene set forth in SEQ ID NO: 29; a region in FGF12 gene set forth in SEQ ID NO: 30; and a region in NPY gene set forth in SEQ ID NO: 31. Also provided are a kit comprising the composition, and use of the composition.

Description

Composition, kit and use for detecting gastric cancer

Cross-references to related applications

This application is based on Chinese patent applications with application number 202210775335.5 and a filing date of July 1, 2022 and an application number 202211430609.3 with a filing date of November 15, 2022, and claims the priority of these Chinese patent applications. The entire contents of these Chinese patent applications are hereby incorporated into this application by reference.

Technical field

The present invention belongs to the field of molecular biology detection, specifically to the field of gastric cancer detection, and more specifically to the detection of methylation levels of gastric cancer gene markers.

Background technique

National malignant tumor statistics released by the National Cancer Center in 2022 show that my country's overall cancer incidence and mortality rates continue to rise, with 4.064 million new cases and 2.414 million deaths. The number of malignant tumors far exceeds that of other countries in the world. It has become one of the major public health issues that seriously threatens the life and health of the Chinese people. Among them, the incidence and mortality of gastric cancer rank third among all cancer types. The number of cases is large, the prognosis is poor, and the prevention and control situation is grim. Studies have shown that the overall prognosis of gastric cancer is closely related to its diagnosis stage, with a long natural history and a slow onset process. Early gastric cancer is limited to the mucosa and submucosa. After surgical treatment, the 5-year survival rate can reach 90%. However, early gastric cancer has no obvious symptoms and is easy to be ignored. Therefore, most gastric cancer cases are usually diagnosed in the middle and late stages, and 5 years after surgery The annual survival rate is low, less than 30%. At present, the treatment costs for stage I, II, III and IV gastric cancer are approximately 50,000-20,000 yuan, 30,000-50,000 yuan, 100,000 yuan/year, and 150,000-200,000 yuan/year respectively. Stomach cancer is difficult and expensive to treat. It can be seen that early screening and early diagnosis of gastric cancer can not only improve the survival rate of patients, but also reduce national and individual medical expenditures. out of burden. How to detect gastric cancer in its early stages and achieve true early screening and early intervention for gastric cancer is an urgent and arduous task.

At present, traditional methods for early screening of gastric cancer mainly include gastroscopy, imaging examinations, and serological examinations, but they generally have certain limitations. Gastroscopy and endoscopic biopsy are the gold standard for gastric cancer diagnosis and can detect gastric cancer earlier. However, it is an invasive examination with poor patient compliance and high professional requirements for endoscopists. At the same time, there is an appointment cycle due to insufficient clinical matching resources. It has a long pain point, so it is difficult to popularize widely. However, imaging examination methods such as X-ray or CT scan have a low diagnostic rate for early gastric cancer and usually carry risks such as radiation and contrast agent safety. Serological screening methods are easy to operate and are non-invasive tests. Currently commonly used markers include PG, G-17, CEA, CA199, etc. However, the sensitivity and specificity of these markers for detecting gastric cancer are low, and their diagnostic value for early screening is low. limited.

In recent years, based on molecular diagnostic technologies such as PCR and NGS, liquid biopsy has continued to develop and make breakthroughs, and has become a powerful weapon for accurate early cancer screening. Judging from the signal abundance and signal intensity of the marker, ctDNA methylation is an ideal detection marker. Research shows that increased methylation levels of tumor suppressor genes usually occur in the early stages of cancer and are a hallmark change in early tumor development; in the CpG islands of the human genome, 60-80% of cytosine residues are methylated Modification, DNA methylation has become a common and abundant source of signals in early cancer screening; in addition, different cancer types have their own unique DNA methylation patterns, which are highly organ-specific and can enable tissue traceability. Therefore, ctDNA methylation is a mainstream detection indicator used by early screening companies at home and abroad.

There is an urgent need in this field for a more effective gastric cancer diagnostic product that can enable early diagnosis of gastric cancer to be both highly sensitive and specific, non-invasive and rapid.

Contents of the invention

In view of this, the present invention provides a composition for detecting gastric cancer, which composition includes a detection reagent for detecting the methylation level of the following region:

The region shown in SEQ ID NO:1 in the OTX1 gene;

The region in the ZNF671 gene as shown in SEQ ID NO:2; and

The region in the ELMO1 gene shown in SEQ ID NO:3.

The region shown in SEQ ID NO:1 in the OTX1 gene, the region shown in SEQ ID NO:2 in the ZNF671 gene, and the region shown in SEQ ID NO:3 in the ELMO1 gene are the promoter regions of the respective genes. A sequence of CpG islands.

Specifically, the SEQ ID NO:1 region of the OTX1 (Genbank accession number: NC_000002.12) gene is as follows:

The SEQ ID NO:2 region of the ZNF671 (Genbank accession number: NC_000019.10) gene is as follows:

The SEQ ID NO:3 region of the ELMO1 (Genbank accession number: NC_000007.14) gene is as follows:

A composition for detecting gastric cancer, the composition comprising a detection reagent for detecting methylation levels in the following regions:

The region shown in SEQ ID NO:29 in the KCNA3 gene;

The region in the FGF12 gene as shown in SEQ ID NO:30; and

The region shown in SEQ ID NO:31 in the NPY gene.

The region shown in SEQ ID NO:29 in the KCNA3 gene, the region shown in SEQ ID NO:30 in the FGF12 gene, and the region shown in SEQ ID NO:31 in the NPY gene are the promoter regions of the respective genes. A sequence of CpG islands.

Specifically, the SEQ ID NO:29 region of the KCNA3 (Genbank accession number: NC_000001.11) gene is as follows:

The SEQ ID NO:30 region of the FGF12 (Genbank accession number: NG_051966.1) gene is as follows:

The SEQ ID NO:31 region of the NPY (Genbank accession number: NG_016148.1) gene is as follows:

Using the composition of the present invention, gastric cancer tissue samples can be clinically detected with at least 97.97% sensitivity; clinically, gastric cancer can be detected sensitively and specifically in the early stages of malignant transformation.

In the present invention, "CpG island" is the abbreviation of cytosine (C)-phosphate (p)-guanine (G), which refers to the promoter and exon region of the gene and is rich in CpG dinucleosides. Some regions of acid are 300 to 3000 bp in length.

In some embodiments, using the detection reagent of the present invention, the methylation level of the CpG island on the corresponding gene present in the sample or a sequence on the CpG island can be detected.

In the present invention, a "sample" is a biological sample selected from an individual. Specifically, for example, selected from cell lines, histological sections, tissue biopsies/paraffin-embedded tissues, body fluids, feces, colonic effluent, urine, plasma, serum, whole blood, isolated blood cells, cells isolated from blood , or a combination thereof.

Preferably, the "sample" of the present invention is plasma, that is, free DNA in plasma.

Free DNA in plasma can be used to detect tumors, which has the characteristics of less harm to patients and good specificity. However, due to its extremely low content in plasma, there is a problem of low sensitivity. Using the detection reagent of the present invention, free DNA in plasma can be used as a sample, and clinical detection can be performed with a sensitivity of at least 88.33% and a specificity of 96.67%.

In the present invention, "detection reagent" refers to a reagent for detecting the methylation level of a gene in a sample. Wherein, the methylation level is measured by amplification-sequencing, chip, and methylation fluorescence quantitative PCR.

In some specific embodiments, detection reagents include, but are not limited to, nucleic acid primers and sequencing Tag sequences for measuring methylation levels by amplification-sequencing.

In some specific embodiments, detection reagents include, but are not limited to, chips that are methylation chips having probes that specifically bind to methylated regions. The chip may include, but is not limited to, Agilent's Human CpG Island Microarrays and Human DNA Methylation Microarrays, Illumina's Infinium HumanMethylation27BeadChip, Infinium HumanMethylation450BeadChip and GoldenGate Methylation Assay, and Roche NimbleGen's Human DNA Methylation 2.1M Deluxe Promoter Array, Human DNA Methylation Array, etc., are used to measure methylation levels by chip.

In some specific embodiments, detection reagents include, but are not limited to, nucleic acid primers and nucleic acid probes for measuring methylation levels by methylation fluorescence quantitative PCR.

Further, the detection reagent also includes internal standard primers and internal standard probes.

In a specific embodiment, the target of the internal standard primers and probes is the β-actin gene.

In a preferred embodiment, the target of the internal standard primers and probes is the region shown in SEQ ID NO:4 of the β-actin gene.

The SEQ ID NO:4 region of the β-actin gene (Gene bank accession number: NC_007992.1) is as follows:

When the detection reagent includes a nucleic acid primer and a nucleic acid probe, the detection reagent detects the methylation level of the nucleic acid in the sample through methylation fluorescence quantitative PCR.

In the present invention, "methylation fluorescence quantitative PCR" refers to converting the region to be detected by sulfite conversion or digesting it with a methylation-sensitive restriction endonuclease, and then using primers and probes specifically designed for the detection target. The needle performs fluorescent quantitative PCR detection to obtain the methylation level of the region to be detected.

Furthermore, the above composition may further include other reagents, specifically, for example, Various reagents required for pretreatment or pretreatment of samples. For example, nucleic acid releasing agents for extracting sample nucleic acid, bisulfite or bisulfite used for conversion, etc.

In some specific embodiments, the detection reagents are as shown in Table 1 and Table 2:

Table 1

Table 2

In some specific embodiments, the four fluorescence channels used in the present invention are FAM, HEX, CY5 and ROX channels, but the actual application is not limited to this, and can be any combination of other fluorescence channels; at the same time, different targets It can also correspond to different fluorescence channels, for example, any fluorescence channel can be used as the internal standard detection channel.

Using this preferred embodiment, the detection sensitivity and specificity are improved by about 10% respectively compared to using the remaining primer and probe compositions. Therefore, using this preferred composition can be used clinically with higher accuracy. Sensitivity and better specificity in detecting early gastric cancer further improve the detection accuracy of early gastric cancer.

In a second aspect, the present invention provides the use of the above composition in preparing a kit for detecting gastric cancer.

Further, the present invention provides the use of the above reagent combination in preparing a kit for detecting gastric cancer using plasma free DNA.

In a third aspect, the present invention provides a kit for detecting gastric cancer, which kit includes the composition as described above.

Further, the kit also includes, but is not limited to, at least one of reagents for extracting nucleic acids, reagents for purifying nucleic acids, and bisulfite.

Further, the kit also includes negative samples.

Specifically, negative samples are human genomic DNA that has been verified by sequencing to have no target gene methylation.

Further, the reagents for extracting nucleic acids are reagents for extracting tissue DNA and reagents for extracting plasma free DNA.

Further, the reagent for extracting nucleic acid is a reagent for extracting plasma free DNA.

Further, the kit also includes at least one of dNTPs, Mg ²⁺ , methylation-sensitive restriction enzyme, PCR buffer, and hot-start enzyme.

Further, the kit also includes a GC enhancer.

Further, the methylation-sensitive restriction endonuclease includes at least one of HpaII, HinP1I and HhaI.

Further, the range of the final concentration of each component is as follows: Mg ²⁺ 1～6mM, dNTPs 1～40mM, methylation-sensitive restriction enzyme 0.01～30U, primer 0.1～40μM, probe 0.1～20μM.

Further, the final concentration of Mg ²⁺ is 2-6mM.

Description of drawings

Figure 1 is the detection of methylation of composition 1 of the present invention in 0.05ng/reaction;

Figure 2 shows the detection of no non-specific amplification of composition 1 of the present invention in 20ng/reaction of unmethylated DNA;

Figure 3 is the detection results of the composition 1 of the present invention for detecting gastric cancer tissue and paracancerous tissue;

Figure 4 shows the detection of target gene methylation in composition 2 of the present invention containing 0.5% in 1ng/μL nucleic acid;

Figure 5 shows the detection of no non-specific amplification of composition 2 of the present invention in 20ng/reaction of unmethylated DNA.

Detailed ways

The present invention will be described in detail below with reference to specific implementation modes and examples, from which the advantages and various effects of the present invention will be more clearly presented. Those skilled in the art should understand that these specific implementation modes and examples are used to illustrate the present invention, but not to limit the present invention.

Example 1. Screening of methylated genes

The present invention collects tumor methylation detection data from the TCGA data set of the UCSC Xena website (https://tcga.xenahubs.net) and the GEO database of the National Center for Biotechnology Information (NCBI). Use gastric cancer and control data to perform differential analysis, and annotate the physical location and genetic information of the differential sites. In order to ensure that the screened fragments have consistent methylation levels, the screening of methylated gene fragments needs to meet the following requirements: 1) It is required that the selected gene fragments have no less than 2 sites with relatively consistent methylation levels; 2) Perform differential analysis on gastric cancer and para-cancerous tissues or normal control tissues, and select gastric cancer samples with high consistency and those with para-cancerous or normal control tissues. There are highly differentially methylated gene segments in normal tissues; 3) Use the whole blood methylation detection data of gastric cancer and healthy samples to perform differential analysis to select differentially hypermethylated gene segments in gastric cancer; 4) Finally, conduct methylation position-by-cell analysis. point analysis to derive candidate methylation sites.

Finally, CpG islands in the promoter regions of three genes, OTX1, ZNF671, and ELMO1, were determined. And the CpG island in the promoter region of the three genes KCNA3, FGF12, and NPY is the optimal combination of detection targets, and the β-actin gene is used as the internal standard for detection. The compositions used in the present invention are shown in Table 1 and Table 2, and the compositions of comparative examples are shown in Table 8 and Table 10.

Example 2. Detection of gene methylation levels in clinical samples

Positive sample: a mixture of standard methylated human genomic DNA and target gene unmethylated human genomic DNA, with a concentration of 1ng/μL containing 10% standard methylated human genomic DNA; a concentration of 1ng/μL containing 1% standard Methylated human genomic DNA; concentration is 1ng/μL containing 0.5% standard methylated human genomic DNA;

Negative sample: human genomic DNA with no target gene methylation verified by sequencing, 2ng/μL.

Testing process:

PCR system configuration: Prepare the PCR reaction solution according to the reagent formulas in Table 3 and Table 4 below;

Methylation-sensitive restriction enzymes in Table 3 include HpaII, HinP1I, and HhaI;

Methylation-sensitive restriction enzymes in Table 4 include HpaII and HinP1I.

table 3

Table 4

Add the sample to the PCR reaction tube at a rate of 10 μL/reaction, then add 40 μL of PCR reaction solution in sequence, cover the PCR tube, shake to mix, and centrifuge briefly for 5 seconds.

Fluorescence PCR reaction and result analysis

1) Place the PCR reaction tube into the sample slot of the amplification instrument and set the name of the sample to be tested in the corresponding order.

2) Fluorescence detection channel selection: OTX1 and KCNA3 select ROX channel (Reportere:ROX,Quencher:None) for detection; ZNF671 and FGF12 select FAM channel (Reportere:FAM,Quencher:None) for detection; ELMO1 and NPY select HEX channel (Reportere: HEX,Quencher:None) detection; β-actin selects CY5 channel (Reportere:CY5,Quencher: None) as internal standard to detect housekeeping genes;

3) Fluorescence quantitative PCR reaction conditions are as shown in Table 5:

table 5

4) Result analysis

After the reaction is completed, the instrument automatically saves the results. You can use the software that comes with the instrument for automatic analysis (you can also manually adjust the starting value, end value and threshold line value of the baseline for analysis). The intersection of the amplification curve and the threshold line is called Ct (cycle threshold, refers to the cycle value experienced when the fluorescence signal in the PCR reaction tube reaches the set threshold). The sensitivity of the detection reagent provided by the invention can reach 0.05ng/reaction (Figures 1 and 4), and there is no non-specific amplification in 20ng/reaction of unmethylated DNA (Figures 2 and 5).

Example 3. Detection of methylation levels of clinical samples using the reagent combination of the present invention

150 clinical plasma samples were collected, including 60 plasma samples from gastric cancer patients, 60 plasma samples from healthy people, and 30 plasma samples from other cancer patients; 150 paraffin section samples of cancer tissue from gastric cancer patients were also collected. Use the Shengxiang Bio plasma free DNA extraction kit to extract plasma cfDNA; use the Sheng Xiang Bio S1008 nucleic acid extraction kit to extract paraffin tissue samples; follow the process of Example 2, use the compositions shown in Table 1 and Table 2 respectively in the same reverse Enzyme digestion of nucleic acid samples and PCR amplification testing should be carried out in the tube. The amplified Ct values of the three target genes in gastric cancer tissue were all less than 32, indicating positive methylation. The amplified Ct values of the three target genes in gastric cancer tissue were significantly different from those of adjacent tissue, indicating that the target had good specificity (Figure 3).

The detection specificity and sensitivity of plasma samples of the composition of the present invention (Table 1) are 96.67% and 88.33% respectively, and the detection sensitivity of tissue samples is 100%. The specific detection results are shown in Table 6 below. It can also be seen from the table that the specificity of the composition of the present invention is relatively good. Only two of the 30 patients with other cancers tested positive.

Table 6

Tissue sample detection sensitivity (%) = number of positive detections/total number of positive cases * 100% = 100%;
Plasma sample detection sensitivity (%) = number of positive detections/total number of positive cases * 100% = 88.33%;
Plasma sample detection specificity (%) = total number of negative detections/total number of negative samples
*100%=96.67%;
Cancer type specificity (%) = number of negative detections in non-gastric cancer patients/total number of non-gastric cancer patients
*100%=93.33%,

The negative samples are the combined samples of all non-gastric cancer patients.

The detection specificity and sensitivity of plasma samples of the composition of the present invention (Table 2) were 95.56% and 86.67% respectively, and the detection sensitivity of tissue samples was 97.97%. The specific detection results are shown in Table 7 below. It can also be seen from the table that the specificity of the composition of the present invention is good, and only 1 of 30 patients with other cancers tested positive.

Table 7


Tissue sample detection sensitivity (%) = number of positive detections/total number of positive cases * 100% = 97.97%;
Plasma sample detection sensitivity (%) = number of positive detections/total number of positive cases * 100% = 86.67%;
Plasma sample detection specificity (%) = total number of negative detections/total number of negative samples
*100%=95.56%;
Cancer type specificity (%) = number of negative detections in non-gastric cancer patients/total number of non-gastric cancer patients
*100%=96.67%,

The negative samples are the combined samples of all non-gastric cancer patients.

Comparative Example 1. Results of testing clinical samples using the comparative example composition of the present invention

150 clinical plasma samples were collected, including 60 plasma samples from gastric cancer patients, 60 plasma samples from healthy people, and 30 plasma samples from other cancer patients; 150 paraffin section samples of cancer tissue from gastric cancer patients were also collected. Use the Shengxiang Bio plasma free DNA extraction kit to extract plasma cfDNA; use the Sheng Xiang Bio S1008 nucleic acid extraction kit to extract paraffin tissue samples; follow the process of Example 2, use the composition shown in Table 8 in the same reaction tube Enzyme digestion of nucleic acid samples and PCR amplification testing are performed. The detection specificity and sensitivity of plasma samples of the comparative composition of the present invention were 77.78% and 70.00% respectively, and the detection sensitivity of tissue samples was 80.67%. The specific detection results are shown in Table 9 below.

Table 8

The OTX1 detection sequence (SEQ ID NO:26) is as follows:

The ZNF671 detection sequence (SEQ ID NO:27) is as follows:

The ELMO1 detection sequence (SEQ ID NO:28) is as follows:

Table 9

Example 4. Results of testing clinical samples using composition 3 of the present invention

150 clinical plasma samples were collected, including 60 plasma samples from gastric cancer patients, 60 plasma samples from healthy people, and 30 plasma samples from other cancer patients; 150 paraffin section samples of cancer tissue from gastric cancer patients were also collected. Use the Shengxiang Bio plasma free DNA extraction kit to extract plasma cfDNA; use the Sheng Xiang Bio S1008 nucleic acid extraction kit to extract paraffin tissue samples; follow the process of Example 2, use the composition shown in Table 10 in the same reaction tube Enzyme digestion of nucleic acid samples and PCR amplification testing are performed.

The detection specificity and sensitivity of plasma samples of the composition of the present invention are 83.33% and 71.67% respectively, and the detection sensitivity of tissue samples is 81.08%. The specific detection results are shown in Table 11 below.

Table 10

Table 11

Claims (10)

A composition for detecting gastric cancer, wherein the composition includes a detection reagent for detecting the methylation level of the following region: The region shown in SEQ ID NO:1 in the OTX1 gene; The region in the ZNF671 gene as shown in SEQ ID NO:2; and The region shown in SEQ ID NO:3 in the ELMO1 gene; or The region shown in SEQ ID NO:29 in the KCNA3 gene; The region in the FGF12 gene as shown in SEQ ID NO:30; and The region shown in SEQ ID NO:31 in the NPY gene. The composition according to claim 1, wherein the detection reagent is a detection reagent used in amplification-sequencing, chip, or methylation fluorescence quantitative PCR to detect methylation level. The composition according to claim 2, wherein the detection reagent is any one or more of nucleic acid primers, sequencing Tag sequences, methylation chips, and nucleic acid probes. The composition according to claim 3, wherein the detection reagent is a nucleic acid primer and a nucleic acid probe. The composition according to claim 4, wherein the nucleic acid primers and nucleic acid probes are: Nucleic acid primers and probes shown in SEQ ID NO:5～13; or Nucleic acid primers and probes shown in SEQ ID NO:32～40. The composition according to claim 5, wherein the detection reagent further includes an internal standard upstream primer, an internal standard downstream primer and an internal standard probe for monitoring. Use of the composition according to any one of claims 1 to 6 in preparing a kit for detecting gastric cancer. A kit for detecting gastric cancer, wherein the kit includes claims 1 to 6 any one of the compositions. The kit according to claim 8, wherein the kit further includes at least one of dNTPs, Mg ²⁺ , methylation-sensitive restriction enzyme, PCR buffer, hot-start enzyme and GC enhancer . The kit according to claim 9, wherein the methylation-sensitive restriction enzyme includes at least one of HpaII, HinP1I and HhaI.