CN116064806B - Composition for evaluating early gastric cancer lymph node metastasis risk and application thereof - Google Patents
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Abstract
The invention discloses a composition for evaluating early gastric cancer lymph node metastasis risk and application thereof, wherein the composition is used for detecting methylation levels of promoter regions of 8 genes such as CLGN, ZNF354C, LMO3, LDHB, ALDH1L1, PTPN13, PPP2R2B, EYA4 and the like in early gastric cancer tissues, evaluating EGC lymph node metastasis risk, helping clinical EGC risk classification so as to evaluate whether Endoscopic Submucosal Dissection (ESD) is performed; the kit has high detection sensitivity, good specificity and high clinical compliance, and has positive significance for EGC risk classification and treatment.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a composition for evaluating early gastric cancer lymph node metastasis risk and application thereof.
Background
Gastric cancer is a clinically common malignancy, and early gastric cancer patients have a good prognosis compared to progressive gastric cancer. Early Gastric Cancer (EGC) refers to the restriction of cancerous tissue to gastric mucosal and submucosal layers, regardless of lesion size and the presence or absence of lymph node metastasis. The survival rate of early gastric cancer patients after surgery (or endoscopic excision) can reach 90-95% in 5 years, and the survival rate of lymph node metastasis in 5 years is 80-85%. Therefore, lymph node metastasis is an important factor in judging prognosis and evaluating treatment regimen, and has better effect on early gastric cancer with lesions localized in mucosa. The current methods for assessing EGC lymph node metastasis are: 1. evaluation according to clinical pathological factors; tumor infiltration depth, tumor size, histological type, etc. are all independent risk factors for EGC lymph node metastasis. 2. Evaluation based on imaging and molecular markers: abdominal ultrasound, CT, MRI, PET, etc., vascular Endothelial Growth Factor (VEGF), p53 gene, etc., are closely related to lymph node metastasis of EGC.
In recent years, with the maturation of DNA methylation detection technology, DNA methylation detection has gradually become a means of clinical auxiliary assessment of cancer treatment, prognosis prediction, and recurrence and metastasis at an early stage of cancer occurrence. The MSP detection technology combines the advantages of fluorescence PCR sensitivity, methylation template specific amplification and the like, is a detection technology with high acceptance in the current clinical test, and has been widely applied to scientific research and clinical detection. Compared with the traditional evaluation method, the evaluation of the lymph node metastasis risk through the gene methylation level has the advantages of low subjective interference, low detection cost, accurate result, strong stability and the like. However, there is currently no technical product on this technology platform that is capable of rapid, specific detection of EGC-related gene methylation.
In order to further improve the evaluation value of the EGC lymph node metastasis risk, the research and development of a molecular detection product which is more specific, effective, more convenient and easier to apply to clinic has important significance.
Disclosure of Invention
In view of the above-mentioned prior art blank, an object of the present invention is to provide a composition for assessing the risk of lymph node metastasis from early gastric carcinoma and its use.
The technical scheme for solving the technical problems is as follows:
the first aspect of the invention provides the use of a combination of methylation levels of eight genes, namely CLGN, ZNF354C, LMO3, LDHB, ALDH1L1, PTPN13 and PPP2R2B, EYA, in the vicinity of the promoter as a target in the preparation of a product for evaluating early stage gastric cancer lymph node metastasis risk;
the application of the combination of the sequence methylation of the promoter nearby regions of eight genes of human CLGN, ZNF354C, LMO, LDHB, ALDH1L1, PTPN13 and PPP2R2B, EYA as targets in the preparation of the early gastric cancer lymph node metastasis risk evaluation product refers to the application of the methylation degree of the eight genes as an auxiliary evaluation index in the preparation of the Endoscopic Submucosal Dissection (ESD) product for evaluating whether the EGC is subjected to the preparation of the Endoscopic Submucosal Dissection (ESD) product.
In a second aspect, the present invention provides a composition for assessing the risk of lymph node metastasis from early gastric carcinoma, comprising:
a. a preparation a for detecting the degree of methylation in at least one target region in the promoter region of the CLGN gene;
b. agent b for detecting the degree of methylation in at least one target region in the promoter region of the ZNF354C gene;
c. agent c for detecting the degree of methylation in at least one target region in the LMO3 gene promoter region;
d. agent d for detecting the degree of methylation in at least one target region in the promoter region of the LDHB gene;
e. agent e for detecting the degree of methylation in at least one target region in the promoter region of the ALDH1L1 gene;
f. a preparation f for detecting the degree of methylation in at least one target region in the promoter region of the PTPN13 gene;
g. a preparation g for detecting the degree of methylation in at least one target region in the promoter region of the PPP2R2B gene;
h. agent h for detecting the degree of methylation in at least one target region in the promoter region of the EYA4 gene.
The above-mentioned promoter region is not strictly limited to a region located within the promoter fragment, but is a gene fragment in the vicinity of the promoter.
Further, the preparation a comprises a primer pair a, wherein the primer pair a comprises a forward primer and a reverse primer, and the forward primer and the reverse primer are respectively shown as SEQ ID No.1 and SEQ ID No. 2;
the preparation b comprises a primer pair b, wherein the primer pair b comprises a forward primer and a reverse primer, and the forward primer and the reverse primer are respectively shown as SEQ ID No.3 and SEQ ID No. 4;
the preparation c comprises a primer pair c, wherein the primer pair c comprises a forward primer and a reverse primer, and the forward primer and the reverse primer are respectively shown as SEQ ID No.5 and SEQ ID No. 6;
the preparation d comprises a primer pair d, wherein the primer pair d comprises a forward primer and a reverse primer, and the forward primer and the reverse primer are respectively shown as SEQ ID No.7 and SEQ ID No. 8;
the preparation e comprises a primer pair e, wherein the primer pair e comprises a forward primer and a reverse primer, and the forward primer and the reverse primer are respectively shown as SEQ ID No.9 and SEQ ID No. 10;
the preparation f comprises a primer pair f, wherein the primer pair f comprises a forward primer and a reverse primer, and the forward primer and the reverse primer are respectively shown as SEQ ID No.11 and SEQ ID No. 12;
the preparation g comprises a primer pair g, wherein the primer pair g comprises a forward primer and a reverse primer, and the forward primer and the reverse primer are respectively shown as SEQ ID No.13 and SEQ ID No. 14;
the preparation h comprises a primer pair h, wherein the primer pair h comprises a forward primer and a reverse primer, and the forward primer and the reverse primer are respectively shown as SEQ ID No.15 and SEQ ID No. 16.
Further, the composition further comprises a preparation i for detecting the methylation degree in at least one target region in the promoter region of the reference gene COL 2A.
Further, the preparation i comprises a primer pair i, wherein the primer pair i comprises a forward primer and a reverse primer, and the forward primer and the reverse primer are respectively shown as SEQ ID No.17 and SEQ ID No. 18.
Further, the composition also comprises any one or more of the following components: PCR reaction solution, enzyme mixture, positive control and negative control.
Further, the PCR reaction solution comprises a buffer solution and MgCl 2 dUTP and dNTPs; the enzyme mixed solution comprises Taq enzyme and UNG enzyme;
preferably, the PCR reaction solution comprises 10 Xbuffer, 25mM MgCl 2 10mM dUTP and 10mM dNTPs; the Taq enzyme is a hot start Taq enzyme, and the UNG enzyme is uracil-N-glycosylase.
Further, the positive control is selected from any one or several of the following: pseudoviruses containing partial sequences of CLGN, ZNF354C, LMO3, LDHB, ALDH1L1, PTPN13, PPP2R2B, EYA methylation genes, pseudoviruses containing partial sequences of human COL2A genes; for example, pseudoviruses containing CLGN, ZNF354C, LMO, LDHB, ALDH1L1, PTPN13, PPP2R2B, EYA methylation genes may be non-replicating adenoviruses.
Further, the negative control is sterile water.
Specifically, the EGC methylation genes CLGN, ZNF354C, LMO3, LDHB, ALDH1L1, PTPN13, and PPP2R2B, EYA have Gene IDs 1047, 30832, 55885, 3945, 10840, 5783, 5521, and 2070, respectively.
In a third aspect the invention provides the use of the composition in the manufacture of a product for assessing the risk of lymph node metastasis from early gastric carcinoma.
Further, the assessing the risk of lymph node metastasis from early gastric carcinoma comprises the steps of:
s1, extracting DNA from a sample and converting the DNA to obtain high-purity bis-DNA;
s2, preparing a sample reaction solution, a positive control reaction solution and a negative control reaction solution;
s3, performing PCR amplification reaction on each sample in the step S2 to obtain the amplification cycle number;
s4, obtaining methylation indexes of the detected gastric cancer tissue sample based on a logistic regression equation.
Further, the logistic regression equation is:
H-Index＝4.41+A1*X1+A2*X2+A3*X3+A4*X4+A5*X5+A6*X6+A7*X7+A8*X8；
wherein H-Index represents methylation Index;
a1 to A8 are clinical coefficients of genes CLGN, ZNF354C, LMO3, LDHB, ALDH1L1, PTPN13 and PPP2R2B, EYA respectively;
x1 to X8 are CT differences between the genes CLGN, ZNF354C, LMO3, LDHB, ALDH1L1, PTPN13, PPP2R2B, EYA and the reference gene, respectively.
Further, the clinical coefficients of the genes are as follows:
| gene | Clinical coefficient |
| CLGN | A1＝-1.27 |
| ZNF354C | A2＝-0.23 |
| LMO3 | A3＝-0.92 |
| LDHB | A4＝1.11 |
| ALDH1L1 | A5＝0.63 |
| PTPN13 | A6＝1.54 |
| PPP2R2B | A7＝0.81 |
| EYA4 | A8＝-1.52 |
Furthermore, the lymph node metastasis risk of the early gastric carcinoma patient can be evaluated according to the H-Index value, and the H-Index threshold value is 3.57, which means that when H-Index is less than or equal to 3.57, the lymph node metastasis risk of the patient is lower, the patient is suitable for the line Endoscopic Submucosal Dissection (ESD), and when H-Index is more than 3.57, the lymph node metastasis risk of the patient is higher, and the patient is unsuitable for the line ESD. In particular, when the H-Index threshold is 3.57, the specificity and sensitivity of the evaluation of the lymph node metastasis risk can reach 100%, which is significantly higher than that of the evaluation marker using a single gene alone.
The invention has the beneficial effects that:
(1) The composition provided by the invention can detect EGC methylation genes CLGN, ZNF354C, LMO3, LDHB, ALDH1L1, PTPN13 and PPP2R2B, EYA4 at the same time, and fills the blank of the existing fluorescent quantitative PCR product in the aspect of EGC lymph node metastasis risk assessment;
(2) The composition provided by the invention has the advantages of high sensitivity, good specificity, strong repeatability, rapid and objective detection result, cost saving and the like in practical application, and has great application prospect in the field of in-vitro diagnosis of gastric cancer methylation.
(3) The composition kit provided by the invention is simple and convenient to operate, can effectively prevent pollution, has the PCR fluorescence detection time (from sample treatment) of only 3-4 hours, can realize full-closed operation in PCR fluorescence detection, and avoids pollution because a tube cover is not required to be opened after the nucleic acid extract of a sample to be detected is added; meanwhile, UNG enzyme is added into the reaction liquid, so that the pollution of amplified products is effectively prevented.
Drawings
Fig. 1 shows DMR heatmaps associated with 53 genes: the invention adopts the DMR analysis results of 4 LNN samples and 4 LNP samples mined in the TCGA database in the early stage:
FIG. 2 shows a box line diagram of methylation delta CT of 130 LNN samples and 100 LNP samples detected by the kit of the invention;
FIG. 3 shows a ROC graph of 230 samples tested using the kit of the invention;
FIG. 4 shows a graph of the H-Index change and corresponding about the Index for detection by the kit of the present invention.
Detailed Description
Other advantages and effects of the present invention will become apparent to those skilled in the art from the following disclosure, which describes the embodiments of the present invention with reference to specific examples. The invention may be practiced or carried out in other embodiments that depart from the specific details, and the details of the present description may be modified or varied from the spirit and scope of the present invention. It should be understood that the process equipment or devices not specifically identified in the examples below are all conventional in the art. Furthermore, it is to be understood that the reference to one or more method steps in this disclosure does not exclude the presence of other method steps before or after the combination step or the insertion of other method steps between these explicitly mentioned steps, unless otherwise indicated; it should also be understood that the combined connection between one or more devices/means mentioned in the present invention does not exclude that other devices/means may also be present before and after the combined device/means or that other devices/means may also be interposed between these two explicitly mentioned devices/means, unless otherwise indicated. Moreover, unless otherwise indicated, the numbering of the method steps is merely a convenient tool for identifying the method steps and is not intended to limit the order of arrangement of the method steps or to limit the scope of the invention in which the invention may be practiced, as such changes or modifications in their relative relationships may be regarded as within the scope of the invention without substantial modification to the technical matter.
Example 1: detection kit and use method thereof
The kit comprises the following components:
PCR reaction liquid, enzyme mixed liquid, detection reaction liquid, positive control and negative control;
the PCR reaction solution comprises 10 Xbuffer, 25mM MgCl2, 10mM dUTP and 10mM dNTPs; the enzyme mixed solution comprises Taq enzyme and UNG enzyme, and the working concentration of the upstream and downstream primers of EGC methylation genes CLGN, ZNF354C, LMO3, LDHB, ALDH1L1, PTPN13 and PPP2R2B, EYA in the detection reaction solution is 10uM.
The nucleotide sequences of the primers for the EGC methylation genes CLGN, ZNF354C, LMO, LDHB, ALDH1L1, PTPN13, PPP2R2B, EYA4 used in this example are shown in Table 1.
TABLE 1 nucleotide sequence listing of EGC marker combination methylation primers in the present invention
Note that: primers were synthesized by specialized synthesis companies using methods conventional in the art.
Positive control: comprises the following components (all known gene sequences):
component (1): is a pseudovirus containing a target sequence, and is a non-replicating adenovirus containing a methylation part sequence of a CLGN gene;
component (2): is a pseudovirus containing a target sequence, and is a non-replicating adenovirus containing a ZNF354C gene methylation part sequence;
component (3): is a pseudovirus containing a target sequence, and is a non-replicating adenovirus containing a LMO3 gene methylation partial sequence;
component (4): the pseudovirus containing target sequence is non-replicating adenovirus containing LDHB gene methylation partial sequence;
component (5): the pseudovirus containing the target sequence is a non-replicating adenovirus containing the ALDH1L1 gene methylation part sequence;
component (6): is a pseudovirus containing a target sequence, and is a non-replicating adenovirus containing a PTPN13 gene methylation partial sequence;
component (7): is a pseudovirus containing a target sequence, and is a non-replicating adenovirus containing a PPP2R2B gene methylation partial sequence;
component (8): is a pseudovirus containing a target sequence, and is a non-replicating adenovirus containing a methylation part sequence of EYA4 genes;
component (9): the pseudovirus containing partial human COL2A gene sequence is non-replicating adenovirus with partial human COL2A gene methylation partial sequence;
negative controlIs sterile water.
Use of a kit
1. The sample is extracted, processed and stored as follows:
1. cell DNA is extracted from the FFPE sample of early gastric cancer. After extraction, the sample concentration and OD260/280 were measured using a Nanodrop-300 micro-spectrophotometer, with OD260/280 between 1.8 and 2.0.
2. The extraction of the cell DNA is completed, the extracted DNA is subjected to bisulfite conversion, unmethylated cytosine (C) is converted into uracil (U), and methylated cytosine (C) is unchanged. Purified bis-DNA was obtained.
The sample should be detected in time after treatment, or stored at-20deg.C, the shelf life is generally not more than 4 months, and long-term storage is carried out at-70deg.C. The sample is prevented from repeated freeze thawing, and is transported at low temperature by dry ice or ice bags.
2. Detection method
1. Reagent preparation (reagent preparation area)
TABLE 2 preparation of reaction solution 1 (sample detection)
| Reaction liquid component | Addition (μl/reaction) |
| PCR reaction solution | 5 |
| Detection of multiple reaction solutions | 5 |
| Enzyme mixed solution | 5 |
| Total volume of | 15 |
TABLE 3 preparation of reaction solution 2 (negative control)
| Reaction liquid component | Addition amount (μl)/per reaction |
| PCR reaction solution | 5 |
| Detection of multiple reaction solutions | 5 |
| Enzyme mixed solution | 5 |
| Total volume of | 15 |
TABLE 4 preparation of reaction solution 3 (positive control)
| Reaction liquid component | Addition amount (μl)/per reaction |
| PCR reaction solution | 5 |
| Detection of multiple reaction solutions | 5 |
| Enzyme mixed solution | 5 |
| Total volume of | 15 |
2. Sample addition
5 mu L of the nucleic acid sample to be detected is added into the PCR amplification tube, the final volume is 20 mu L per tube, the tube cover is closed, and the instant low-speed centrifugation is carried out, so that the detection is carried out on a PCR instrument.
PCR amplification (amplification Chamber)
TABLE 5 amplification cycle parameter settings
And after the setting is finished, storing a file and running a reaction program.
Example 2: detection accuracy test of kit
This experimental example groups early gastric cancer FFPE samples into Lymph Node Negative (LNN) samples (n=130), lymph Node Positive (LNP) samples (n=100) according to clinical pathology and imaging diagnosis, and the early gastric cancer FFPE samples were experimentally detected by using the kit and experimental method provided in example 1.
As shown in fig. 2, the methylation level of the 8 genes of interest was significantly lower in the LNN group than in the LNP group.
Example 3: combination of kit markers methylation level and ROC curve for EGC lymph node metastasis
The experimental example provides an ROC curve for detecting methylation level and lymph node metastasis risk by using an early gastric cancer FFPE DNA multi-target detection technology.
The experimental example carries out experimental detection on the FFPE sample of the early gastric cancer by using the kit and the experimental method provided in the embodiment 1.
As shown in FIG. 3, the early gastric cancer FFPE sample DNA polygene detection technology can obviously distinguish LNN from LNP, and the Area (AUC) of the combined detection ROC curve of the kit marker combination is 1 (0.95-0.98, 95% CI; P < 0.0001).
Example 4: logistic regression analysis
And obtaining a logistic regression equation for judging early gastric cancer lymph node metastasis conditions through experiments: h-index=4.41+a1x1+a2x2+a3x3+a4x4+a5 x5+a6 x6+a7 x7+a8x8; wherein A1-A8 are clinical coefficients, and the clinical coefficient results are shown in Table 7; the differences in the amplification cycle numbers of CLGN, ZNF354C, LMO3, LDHB, ALDH1L1, PTPN13, PPP2R2B, EYA4 and the internal reference gene COL2A were X1, X2, X3, X4, X5, X6, X7, X8, respectively. And (3) injection: the difference formula of the amplification cycle number is X= CT (target gene) -CT (COL 2A)
TABLE 7 clinical coefficients
The sensitivity and specificity of the joint detection of early gastric cancer lymph node metastasis by analyzing methylation fitting regression analysis of 8 genes are obviously higher than those of detection analysis results of single genes.
In conclusion, the marker combination methylation detection result provided by the invention is accurate and reliable; by applying the kit provided by the invention, early gastric cancer cases can be conveniently and rapidly screened, a new calculation method is communicated, a detection conclusion can be accurately obtained, EGC risk classification is assisted in clinic, and a subsequent treatment scheme is evaluated.
The above examples are provided to illustrate the disclosed embodiments of the invention and are not to be construed as limiting the invention. In addition, various modifications, as well as variations in the methods and combinations of markers set forth herein will be apparent to those skilled in the art without departing from the scope and spirit of the invention. While the invention has been specifically described in connection with various specific preferred embodiments thereof, it should be understood that the invention should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in the art are intended to be within the scope of the present invention.
Claims (7)
1. A composition for assessing the risk of lymph node metastasis from early gastric carcinoma, comprising:
a. a preparation a for detecting the methylation degree of the CLGN gene promoter region;
b. a preparation b for detecting the methylation degree of the ZNF354C gene promoter region;
c. agent c for detecting the methylation degree of the LMO3 gene promoter region;
d. a preparation d for detecting the methylation degree of the promoter region of the LDHB gene;
e. agent e for detecting the methylation degree of the promoter region of the ALDH1L1 gene;
f. a preparation f for detecting the methylation degree of the promoter region of the PTPN13 gene;
g. agent g for detecting the methylation degree of the promoter region of the PPP2R2B gene;
h. agent h for detecting the degree of methylation of the EYA4 gene promoter region; the preparation a comprises a primer pair a, wherein the primer pair a comprises a forward primer and a reverse primer, and the forward primer and the reverse primer are respectively shown as SEQ ID No.1 and SEQ ID No. 2;
the preparation b comprises a primer pair b, wherein the primer pair b comprises a forward primer and a reverse primer, and the forward primer and the reverse primer are respectively shown as SEQ ID No.3 and SEQ ID No. 4;
the preparation c comprises a primer pair c, wherein the primer pair c comprises a forward primer and a reverse primer, and the forward primer and the reverse primer are respectively shown as SEQ ID No.5 and SEQ ID No. 6;
the preparation d comprises a primer pair d, wherein the primer pair d comprises a forward primer and a reverse primer, and the forward primer and the reverse primer are respectively shown as SEQ ID No.7 and SEQ ID No. 8;
the preparation e comprises a primer pair e, wherein the primer pair e comprises a forward primer and a reverse primer, and the forward primer and the reverse primer are respectively shown as SEQ ID No.9 and SEQ ID No. 10;
the preparation f comprises a primer pair f, wherein the primer pair f comprises a forward primer and a reverse primer, and the forward primer and the reverse primer are respectively shown as SEQ ID No.11 and SEQ ID No. 12;
the preparation g comprises a primer pair g, wherein the primer pair g comprises a forward primer and a reverse primer, and the forward primer and the reverse primer are respectively shown as SEQ ID No.13 and SEQ ID No. 14;
the preparation h comprises a primer pair h, wherein the primer pair h comprises a forward primer and a reverse primer, and the forward primer and the reverse primer are respectively shown as SEQ ID No.15 and SEQ ID No. 16.
2. The composition according to claim 1, further comprising a preparation i for detecting the methylation degree of the promoter region of the reference gene COL 2A; the preparation i comprises a primer pair i, wherein the primer pair i comprises a forward primer and a reverse primer, and the forward primer and the reverse primer are respectively shown as SEQ ID No.17 and SEQ ID No. 18.
3. The composition of claim 1, further comprising any one or more of the following components: PCR reaction solution, enzyme mixed solution, positive control and negative control; the PCR reaction solution comprises buffer solution and MgCl 2 dUTP and dNTPs; the enzyme mixed solution comprises Taq enzyme and UNG enzyme.
4. The composition of claim 3, wherein the positive control is: pseudoviruses containing the partial sequences of the CLGN, ZNF354C, LMO3, LDHB, ALDH1L1, PTPN13, PPP2R2B, EYA methylation genes, pseudoviruses containing the partial sequences of the human COL2A genes.
5. Use of a composition according to any one of claims 1 to 4 for the preparation of a product for assessing risk of lymph node metastasis from early gastric carcinoma.
6. The use according to claim 5, wherein said assessing the risk of lymph node metastasis from early gastric carcinoma comprises the steps of:
s1, extracting DNA from a sample and converting the DNA to obtain high-purity bis-DNA;
s2, preparing a sample reaction solution, a positive control reaction solution and a negative control reaction solution;
s3, performing PCR amplification reaction on each sample in the step S2 to obtain the amplification cycle number;
s4, obtaining methylation indexes of the detected gastric cancer tissue sample based on a logistic regression equation.
7. The use according to claim 6, wherein the logistic regression equation is:
H-Index＝4.41+A1*X1+A2 *X2+A3*X3+A4*X4+A5*X5+A6*X6+A7*X7+A8*X8；
wherein H-Index represents methylation Index;
A1-A8 are clinical coefficients of genes CLGN, ZNF354C, LMO3, LDHB, ALDH1L1, PTPN13 and PPP2R2B, EYA respectively; X1-X8 are CT differences between genes CLGN, ZNF354C, LMO3, LDHB, ALDH1L1, PTPN13, PPP2R2B, EYA and the reference gene respectively.
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